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Dynamics and Rigidity in an Intrinsically Disordered Protein, ??-Casein
Abstract
The emergence of intrinsically disordered proteins (IDP) as a recognized structural class has forced the community to confront a new paradigm of structure, dynamics, and mechanical properties for proteins. We present novel data on the similarities and differences in the dynamics and nanomechanical properties of IDPs and other biomacromolecules on the picosecond timescale. An IDP, β-casein (CAS), has been studied in a calcium bound and unbound state using neutron and light scattering techniques. We show that CAS partially folds and stiffens upon calcium binding, but in the unfolded state it is softer than folded proteins such as green fluorescence protein (GFP). We also see that some localized diffusive motions in CAS have larger amplitude than in GFP at this timescale, but are still smaller than those observed in tRNA. In spite of these differences, CAS dynamics are consistent with the classes of motions seen in folded protein on this time scale.
... The observation of this process with improved instrumental resolution motivated our Neutron Spin Echo (NSE) and NBS experiments with β-casein in solution in this study. Another motivation was to examine if β-casein in solution and in powder state provide different quasi-elastic neutron scattering spectra in comparison of the literatures by Perticaroli et al. (28) and Dhinsda et al. (29). We ask the question, whether similar dynamic processes are observed or whether the bulk solvent induces additional molecular motions. ...
... We ask the question, whether similar dynamic processes are observed or whether the bulk solvent induces additional molecular motions. The biological and industrial background of natively disordered β-casein has been excellently presented in the literatures (28,29), and is not discussed here again. Instead, we focus on neutron scattering experiments performed with other natively disordered proteins: The human tau-protein and its coupling to hydration water (30) and disordered Myelin Basic Protein studied in solution by NSE (31). ...
... The NSE process of β-casein in aqueous solution, at solvent viscosities of 1 cP and τ1 ≈ 1 ns, yields G∞ ~ 10 9 cP/s (0.001 GPa) about a factor of 100 less than for viscous solvents. G∞ is closely related to the Young's modulus E by 1/3 E < G∞ < ½ E. For dry β-casein powder, a Young's modulus of 7 GPa was determined and 9.7 GPa for the compact β-barrel protein GFP (28). The characteristic density relaxation rate will thus depend on the scale, increasing with q according to ΓD(q) = Dq 2 . ...
β-casein undergoes a reversible endothermic self-association, forming protein micelles of limited size. In its functional state, a single β-casein monomer is unfolded, which creates a high structural flexibility, supposed to play a major role in preventing the precipitation of calcium phosphate particles. We characterize the structural flexibility in terms of nano-second molecular motions, depending on the temperature by quasi-elastic neutron scattering. Our major questions are: Does the self- association reduce the chain flexibility? How does the dynamic spectrum of disordered caseins differ from a compactly globular protein? How does the dynamic spectrum of β-casein in solution differ from that of a protein in hydrated powder states? We report on two relaxation processes on a nano-second and a sub-nano-second time scale for β-casein in solution. Both processes are analyzed by Brownian Oscillator model, by which the spring constant can be defined in the isotropic parabolic potential. The slower process, which is analyzed by neutron spin echo, seems a characteristic feature of the unfolded structure. It requires bulk solvent and is not seen in hydrated protein powders. The faster process, which is analyzed by neutron backscattering, has a smaller amplitude and requires hydration water, which is also observed with folded proteins in the hydrated state. The self-association had no significant influence on internal relaxation, and thus a β-casein protein monomer flexibility is preserved in the micelle. We derive spring constants of the faster and slower motions of β-caseins in solution, and compared them with those of some proteins in various states; folded or hydrated powder.
... * Analysis of secondary structure of β-casein/PS-20 samples was processed by the CAPITO program [20]. In the case of bovine β-casein, the secondary structure content showed 13.5% of α-helical, 17.9% of β-sheet, and 69.1% of irregular conformation (Table 2), indicating the intrinsically disordered structure of bovine β-casein [34]. Upon binding with polysorbate-20, the content of α-helical, β-sheet and irregular conformation was calculated as 16.8%, 21.0%, and 62.7%, respectively, suggesting that the α-helical and β-sheet conformation of bovine β-casein increased, but the irregular structure was reduced. ...
... The effect of increasing molar ratios of caprine casein (αs1-II) on the fluorescence emission spectrum of resveratrol was studied and the results are shown in Figure 4. When excited at its adsorption maximum of 308 nm, resveratrol exhibited a fluorescence emission with a peak at around In the case of bovine β-casein, the secondary structure content showed 13.5% of α-helical, 17.9% of β-sheet, and 69.1% of irregular conformation (Table 2), indicating the intrinsically disordered structure of bovine β-casein [34]. Upon binding with polysorbate-20, the content of α-helical, β-sheet and irregular conformation was calculated as 16.8%, 21.0%, and 62.7%, respectively, suggesting that the α-helical and β-sheet conformation of bovine β-casein increased, but the irregular structure was reduced. ...
... Upon binding with polysorbate-20, the content of α-helical, β-sheet and irregular conformation was calculated as 16.8%, 21.0%, and 62.7%, respectively, suggesting that the α-helical and β-sheet conformation of bovine β-casein increased, but the irregular structure was reduced. The CD data suggest that the changes in the secondary structure of bovine β-casein induced by polysorbate-20 are related to the disordered regions and the hydrophobic domains of this protein [34]. The lauric acid chains of polysorbate-20 may be aligned along the α-helical and β-sheet structures in order to stabilize disordered regions and expose aromatic residues of bovine β-casein, thereby increasing the secondary structure of bovine β-casein [35]. ...
The incorporation of hydrophobic ingredients, such as resveratrol (a fat-soluble phytochemical), in nanoemulsions can increase the water solubility and stability of these hydrophobic ingredients. The nanodelivery of resveratrol can result in a marked improvement in the bioavailability of this health-promoting ingredient. The current study hypothesized that resveratrol can bind to caprine casein, which may result in the preservation of the biological properties of resveratrol. The fluorescence spectra provided proof of this complex formation by demonstrating that resveratrol binds to caprine casein in the vicinity of tryptophan amino acid residues. The caprine casein/resveratrol complex is stabilized by hydrophobic interactions and hydrogen bonds. Hence, to study the rate of resveratrol degradation during processing/storage, resveratrol losses were determined by reversed-phase high performance liquid chromatography (RP-HPLC) in nanoemulsions stabilized by bovine and caprine caseins individually and in combination with polysorbate-20. At 48 h oxidation, 88.33% and 89.08% was left of resveratrol in the nanoemulsions stabilized by caprine casein (αs1-I)/polysorbate-20 complex and caprine (αs1-II)/polysorbate-20 complex, while there was less resveratrol left in the nanoemulsions stabilized by bovine casein/polysorbate-20 complex, suggesting that oxygen degradation was involved. The findings of this study are crucial for the food industry since they imply the potential use of caprine casein/polysorbate-20 complex to preserve the biological properties of resveratrol.
... To verify this hypothesis, changes in glycation behavior of bovine serum albumin (BSA, a heart-shaped folded protein), β-Lactoglobulin (β-Lg, a rigid globular protein) and β-casein (β-CN, an open and intrinsic disordered protein) after PT were compared in this work (Bhattacharya, Jain, & Mukhopadhyay, 2011;Perticaroli, Nickels, Ehlers, Mamontov, & Sokolov, 2014;Qin et al., 1998). In addition to measuring common glycation parameters, including loss of free amino groups and increases in UV absorbance, we also analyzed the changes in glycation sites by using proteomics method and aggregation behavior of the protein by using transmission electron microscope (TEM) to further reveal the underlying mechanism. ...
... By contrast, the particle size of β-CN almost remained constant in 60 min of PT. The intrinsic disordered structure of β-CN renders it highly stable, which could account for the relative stable particle size of β-CN during 60 min of PT (Perticaroli et al., 2014). ...
... Variations in k values were observed among β-CN samples in Table 1, whereas the A values of all samples were similar to each other. In addition, the glycation parameters in Table 2 show no significant differences except for UV absorbance at 294 nm and 420 nm in β-CN with 40 min of PT. β-CN has been widely reported to have a highly flexible structure and can aggregate into incompact micelles (Perticaroli et al., 2014;Ossowski et al., 2012). The flexible and open structure of β-CN may minimize the effect of unfolding behavior induced by PT on the following glycation process. ...
Heating is the most commonly used treatment in food industry. This work reported the influence of preheating treatment (PT, 98 °C) on the following glycation behavior of bovine serum albumin (BSA), β-lactoglobulin (β-Lg) and β-casein (β-CN) at 90 °C. The PT was shown to induce both the unfolding and aggregation of proteins, which changed their subsequent glycation kinetics based on measurements of free amino group content and UV absorbance at 294 and 420 nm. Ten minutes of PT unfolded the globular structures of BSA and β-Lg and exposed more glycation sites, which largely up-regulated their glycation degrees. By contrast, 40 min of PT induced more severe aggregation in BSA and β-Lg during PT and the subsequent glycation process, which hid a portion of glycation sites and down-regulated their glycation degree. The glycation behavior of β-CN was almost not affected by PT, possibly due to its intrinsic disordered structure and high thermal stability. Proteomics analysis showed that PT resulted in changes in the number of glycated structures and preference of lysine residues to be glycated. These results suggested PT as an effective way in regulating the glycation behavior of dietary proteins, depending on PT duration and structure of treated protein.
... These findings suggested that glycation derived from α-dicarbonyl considerably affected the size and conformation of β-Lg aggregates, possibly due to steric hindrance and electrostatic interactions, as reported previously (Liu & Zhong, 2012;Pinto et al., 2012). As comparison, aggregation behavior of β-CN was affected to lesser degree, possibly because of its intrinsically disordered structure (Perticaroli, Nickels, Ehlers, Mamontov, & Sokolov, 2014) which weakened the effect of glycation. ...
... For example, GO-derived glycation led to a 3.2% reduction in the DH of β-CN in the intestinal stage, compared to a 16.4% reduction in that of β-Lg. As discussed previously, β-CN is an intrinsically disordered protein that maintains its flexible structure during heating (Perticaroli et al., 2014). The open and flexible structure of β-CN may weaken the influence of glycation-induced steric hindrance or other forms of repulsive effects on the action of digestive proteases. ...
... However, all of these aggregates in glycated β-CN appeared to be degraded during gastric digestion and almost disappeared immediately in the subsequent intestinal digestion. These discrepancies are attributable to the open and flexible structure of β-CN (Perticaroli et al., 2014), which weakens the hindrance effect of glycation-induced crosslinked structure on the action of digestive proteases. Because of the decreased digestibility mediated by glycation, as shown by DH and the SDS-PAGE, more digests with larger sizes were found in the glycated samples (Fig. 4, B and C). ...
α-Dicarbonyl compounds, which are widely found in common consumed food, are one of the precursors of advanced glycation end products (AGEs). In this study, the effect of glycation derived from glyoxal (GO), methylglyoxal (MGO) or butanedione (BU) on the in vitro digestibility of β-casein (β-CN) and β-lactoglobulin (β-Lg) was investigated. Glycation from α-dicarbonyl compounds reduced the in vitro digestibility of studied proteins in both gastric and intestinal stage. In addition, glycation substantially altered the peptides released through gastric and gastrointestinal digestion, as detected by liquid chromatography electrospray-ionization tandem mass spectrometry (LC-ESI-MS/MS). Crosslinked glycation structures derived from BU considerably reduced the sensitivity of glycated β-Lg towards digestive proteases, albeit to a lesser degree in glycated β-CN due to its intrinsic unordered structure. By contrast, non-crosslinked AGEs that formed adjacent to enzymatic cleavage sites did not block the enzymatic reaction in several cases, as evidenced by the corresponding digested peptides modified with glycation structures. These findings expand our understanding of the nutritional influence of α-dicarbonyl compounds and health impact of relevant dietary AGEs.
... These findings suggested that glycation derived from α-dicarbonyl considerably affected the size and conformation of β-Lg aggregates, possibly due to steric hindrance and electrostatic interactions, as reported previously (Liu & Zhong, 2012;Pinto et al., 2012). As comparison, aggregation behavior of β-CN was affected to lesser degree, possibly because of its intrinsically disordered structure (Perticaroli, Nickels, Ehlers, Mamontov, & Sokolov, 2014) which weakened the effect of glycation. ...
... For example, GO-derived glycation led to a 3.2% reduction in the DH of β-CN in the intestinal stage, compared to a 16.4% reduction in that of β-Lg. As discussed previously, β-CN is an intrinsically disordered protein that maintains its flexible structure during heating (Perticaroli et al., 2014). The open and flexible structure of β-CN may weaken the influence of glycation-induced steric hindrance or other forms of repulsive effects on the action of digestive proteases. ...
... However, all of these aggregates in glycated β-CN appeared to be degraded during gastric digestion and almost disappeared immediately in the subsequent intestinal digestion. These discrepancies are attributable to the open and flexible structure of β-CN (Perticaroli et al., 2014), which weakens the hindrance effect of glycation-induced crosslinked structure on the action of digestive proteases. Because of the decreased digestibility mediated by glycation, as shown by DH and the SDS-PAGE, more digests with larger sizes were found in the glycated samples (Fig. 4, B and C). ...
This work reports the influence of GO-derived glycation on the gastrointestinal enzymatic hydrolysis of β-lactoglobulin and β-casein. Reduced digestibility of glycated proteins was found in both gastric and intestinal stage. Distribution of Maillard reaction products (MRPs) in digests with different molecular weight ranges was investigated subsequently. The colorless and brown MRPs largely presented in the digests smaller than 20 kDa. However, the resistance of fluorescent AGEs to enzymatic hydrolysis gradually increased during glycation, rendering fluorescent AGEs largely present in the digests larger than 20 kDa. No free N (ε)-carboxymethyllysine (CML) was detected in digests. The relative amount of CML in digests larger than 1 kDa was higher than that of Lys, demonstrating the hindrance of CML to enzymatic hydrolysis. This study highlights the resistance of GO-derived AGEs to digestive proteases via blockage of tryptic cleavage sites or steric hindrance, which is a barrier to the absorption of dietary AGEs.
... 38,39 Both folded proteins and intrinsically disordered proteins (dry or hydrated) appear to exhibit the same classes of motions at these time scales, with similar response to changes in temperature and hydration level. 42 There is a pronounced increase in the spectra of the α-elastin hydrogel associated with water enhanced, localized diffusive motions below ∼1 meV, relative to both the globular protein green fluorescent protein (GFP) and the unfolded protein βcasein (CAS) hydrated powders. 41,42 These motions in the elastin hydrogel show a pronounced maximum around ∼0.3 meV at 300 K (∼2.2 ps). ...
... 42 There is a pronounced increase in the spectra of the α-elastin hydrogel associated with water enhanced, localized diffusive motions below ∼1 meV, relative to both the globular protein green fluorescent protein (GFP) and the unfolded protein βcasein (CAS) hydrated powders. 41,42 These motions in the elastin hydrogel show a pronounced maximum around ∼0.3 meV at 300 K (∼2.2 ps). The increased water content in the elastin hydrogel (h = 1) relative to CAS and GFP hydrated powders (h = 0.4) 41,42 enhances the localized diffusion of the protein which is responsible for the larger scattered intensity. ...
... The fraction of methyl protons was determined from the amino acid sequence of elastin 44 by taking the fraction of nonexchangeable hydrogen atoms found on methyl groups (P M = 0.38). There is a larger fraction of methyl protons in α-elastin (P M = 0.38) relative to GFP (P M = 0.26 41 ) and CAS (P M = 0.29 42 ) accounting for a small source of extra intensity. The radius of the methyl rotations was taken to be R M = 1.03 Å; 45 these values were used at both temperatures. ...
Elastin is a structural protein and biomaterial that provides elasticity and resilience to a range of tissues. This work provides insights into the elastic properties of elastin and its peculiar inverse temperature transition (ITT). These features are dependent on hydration of elastin and are driven by a similar mechanism of hydrophobic collapse to an entropically favorable state. Using neutron scattering, we quantify the changes in the geometry of molecular motions above and below the transition temperature, showing a reduction in the displacement of water-induced motions upon hydrophobic collapse at the ITT. We also measured the collective vibrations of elastin gels as a function of elongation, revealing no changes in the spectral features associated with local rigidity and secondary structure, in agreement with the entropic origin of elasticity.
... Gallat et al. [47] showed that the intrinsically disordered tau protein behaves in a more dynamic manner compared with MBP, a globular protein of similar molecular mass. The conclusions, drawn by Gallat and co-workers, that a disordered protein behaves more dynamically then a folded counterpart, have been confirmed by quasi-elastic neutron studies of the intrinsically disordered b-casein [70,71]. ...
... At higher temperatures, the MSD of OPN 1-149 is higher than that of OPN CPN, indicative of increased dynamics of free OPN 1-149. This behaviour is similar to that previously found in dynamics studies of b-casein which stiffens upon binding to calcium ions (although in this case partial folding was also observed in the bound protein) [70]. When comparing CPN-forming phosphopeptide dynamics, however, the number and distribution of clustered regions of phosphorylated residues ( [14]; PC) must be kept in mind. ...
... The dynamical behaviour observed for OPN 1-149 is in agreement with results obtained by other techniques on the intrinsically disordered b-casein, and furthermore agrees well with other EINS fixed window temperature scans performed on the intrinsically disordered Tau protein [70,71]. This suggests that an increased dynamical behaviour, compared to globular proteins, may be a functionally required property of IDPs. ...
The sequestration of calcium phosphate by unfolded proteins is fundamental to the stabilization of biofluids supersaturated with respect to hydroxyapatite, such as milk, blood or urine. The unfolded state of osteopontin (OPN) is thought to be a prerequisite for this activity, which leads to the formation of core–shell calcium phosphate nanoclusters. We report on the structures and dynamics of a native OPN peptide from bovine milk, studied by neutron spectroscopy and small-angle X-ray and neutron scattering. The effects of sequestration are quantified on the nanosecond– ångström resolution by elastic incoherent neutron scattering. The molecular fluctuations of the free phosphopeptide are in agreement with a highly flexible protein. An increased resilience to diffusive motions of OPN is corroborated by molecular fluctuations similar to those observed for globular proteins, yet retaining conformational flexibilities. The results bring insight into the modulation of the activity of OPN and phosphopeptides with a role in the control of biomineralization. The quantification of such effects provides an important handle for the future design of new peptides based on the dynamics–activity relationship.
... 7,8 Our results showed that random coil structures are softer than a-helices, which are in turn softer than b-sheets, 7,8 and that such properties are strongly influenced by electrostatic interactions. 9 While our studies have used non-perturbing scattering techniques, other experimental 10 and theoretical 11 work has built from the pioneering pulling experiments on the protein titin. This work has evolved to include many other natural 12 and synthetic materials with a range of mechanical properties. ...
... This feature occurs on a subpicosecond time scale and appears in both neutron and LS spectra of proteins, polymers, and glass forming systems below $5 meV. [7][8][9][44][45][46][47][48][49] Interpretation of these data is that the higher the frequency of the BP, the stiffer is the material. Rigidity on the length scale of $100 nm was investigated through Brillouin LS measurements which detect propagating sound waves with wavelength of the order of $100 nm and frequencies of $10 GHz. ...
... To establish a quantitative estimate of the boson peak frequency, m BP , for all our samples we fit the spectral densities according to the equation 8,9,45,51 : ...
Poly-L-glutamic acid (PGA) is a widely used biomaterial, with applications ranging from drug delivery and biological glues to food products and as a tissue engineering scaffold. A biodegradable material with flexible conjugation functional groups, tunable secondary structure and mechanical properties, PGA has potential as a tunable matrix material in mechanobiology. Recent studies in proteins connecting dynamics, nanometer length scale rigidity, and secondary structure suggest a new point of view from which to analyze and develop this promising material. We have characterized the structure, topology, and rigidity properties of PGA prepared with different molecular weights and secondary structures through various techniques including SEM, FT-IR, light and neutron scattering spectroscopy. On the length scale of a few nanometers rigidity is determined by hydrogen bonding interactions in presence of neutral species and by electrostatic interactions when the polypeptide is negatively charged. When probed over hundreds of nanometers, the rigidity of these materials is modified by long range intermolecular interactions that are introduced by the supramolecular structure. This article is protected by copyright. All rights reserved.
Copyright © 2015 Wiley Periodicals, Inc., A Wiley Company.
... The hydration level h used in this work was 0.4 g of water per gram of casein (h = 0.4). For this hydration level, proteins are known to be enveloped with a water monolayer covering its surface to maintain its activity [35]; this hydration level is often employed in protein studies by neutron scattering, see e.g., [36,37]. For comparison purpose, we investigated here both hydrated and dry protein samples. ...
... It is worth noting in this respect that the disorder-to-order transition in IDPs is widely recognized as a common property of IDPs upon their interaction with a binding partner [1][2][3][4][5][6][7][8][9]34]. Data of neutron and light scattering techniques [37] show that β-casein partially folds and stiffens upon calcium binding, and that in the unfolded state it is softer Molecules 2021, 26, 5971 7 of 12 than folded proteins-the features analogous to those observed here, with the difference that the transition here is induced by the temperature increase. ...
Intrinsically disordered proteins (IDPs) are proteins that possess large unstructured regions. Their importance is increasingly recognized in biology but their characterization remains a challenging task. We employed field swept Electron Spin Echoes in pulsed EPR to investigate low-temperature stochastic molecular librations in a spin-labeled IDP, casein (the main protein of milk). For comparison, a spin-labeled globular protein, hen egg white lysozyme, is also investigated. For casein these motions were found to start at 100 K while for lysozyme only above 130 K, which was ascribed to a denser and more ordered molecular packing in lysozyme. However, above 120 K, the motions in casein were found to depend on temperature much slower than those in lysozyme. This abrupt change in casein was assigned to an ordering transition in which peptide residues rearrange making the molecular packing more rigid and/or more cohesive. The found features of molecular motions in these two proteins turned out to be very similar to those known for gel-phase lipid bilayers composed of conformationally ordered and conformationally disordered lipids. This analogy with a simpler molecular system may appear helpful for elucidation properties of molecular packing in IDPs.
... Additionally, Sokolov and co-workers indicated that the boson peak provides a good measure of protein softness and rigidity (34)(35)(36). ...
... The spectra above $5 meV are identical for all the samples, as expected by the lower sensitivity of the localized harmonic motions to environment. To evaluate the intrinsic boson peak position and intensity, the spectra were fitted by the traditional expression (34)(35)(36)48,49); ...
The softness and rigidity of proteins are reflected in the structural dynamics, which are in turn affected by the environment. The characteristic low-frequency vibrational spectrum of a protein, known as boson peak, is an indication of the structural rigidity of the protein at a cryogenic temperature or dehydrated conditions. In this article, the effect of hydration, temperature, and pressure on the boson peak and volumetric properties of a globular protein are evaluated by using inelastic neutron scattering and molecular dynamics simulation. Hydration, pressurization, and cooling shift the boson peak position to higher energy and depress the peak intensity and decreases the protein and cavity volumes. We found the correlation between the boson peak and cavity volume in a protein. A decrease of cavity volume means the increase of rigidity, which is the origin of the boson peak shift. Boson peak is the universal property of a protein, which is rationalized by the correlation.
... This finding may be related to the intrinsic disordered structure of -CN. 33 The flexible structure and high thermal stability of -CN may weaken hindrance induced by glycation structures to the action of digestive proteases. ...
... 9 The hindrance of crosslinked glycation structure to proteolysis in this study was shown to be less significant in glycated -CN, which could also be attributed to its intrinsic flexible and open structure. 33 Due to the decreased digestibility shown by fluorescamine assay and SDS-PAGE images, more digests with larger sizes were found in the 10 mmol L −1 glucose-glycated samples (Fig. 1, A4, B4, C4). In gastrointestinal digests of the control -CN, -Lg and BSA (Fig. 1, A3, B3, C3), only a few micelles or fibrils were found. ...
BACKGROUND
Milk proteins are widely used in food production and glycated by reducing sugar in many occasions. Although many works have reported the digestibility of glycated milk protein, most of them have focused on measuring degree of hydrolysis (DH), showing SDS‐PAGE image of digests. Detail information on the changes in peptide composition of digests was seldom revealed. Therefore, in addition to measuring the DH and showing the SGS‐PAGE images of digests, we also analyzed the peptidomics in digests using LC‐ESI‐MS/MS and Mascot database in this work to further reveal the influence of glycation on protein nutrition.
RESULTS
Compared with β‐Lg and BSA, DH of β‐CN was suppressed to lesser extent by glycation in both gastric and intestinal stage. Aggregates of glycated BSA were less sensitive to the action of digestive enzymes throughout the gastrointestinal digestion according to SDS‐PAGE images. Change in peptides compositions of digests induced by glycation were distinctly displayed, showing both absence of peptides and occurrence of new peptides, based on the results obtained from LC‐ESI‐MS/MS.
CONCLUSIONS
Glycation can greatly change the peptides composition in digests of milk protein. The nutritional impact of the change in the peptides composition requires further investigation, and the impact of MRPs in unabsorbed digests on the gut flora should be an interest field for further studies.
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... As illustrated in Table 2, CAH has an intrinsically disordered structure (45.4%) due to a high proline content (Alaimo, Farrell, & Germann, 1999; Bu nka, K rí z, Veli ckov a, Bu nkov a, & Kr a cmar, 2009). This open and flexible structure of CAH could provide abundant space for the modification of the Lys and Arg residues during the glycation, which minimised the influence of glycation on its secondary structure (Perticaroli, Nickels, Ehlers, Mamontov, & Sokolov, 2014), as seen in MRP samples. Adding glycation-inducing agents increased b-sheet (left-twisted, relaxed, and right-twisted) and bturn contents while decreasing the disordered fraction. ...
The effects of the Maillard reaction on the glycation, structural, and functional characteristics of Maillard reaction products (MRPs) of casein acid hydrolysate (CAH) was assessed using reducing sugars (methylglyoxal, glucose, galactose, lactose, and maltose) at two different incubation conditions- 6 h, 80º C and 8 h, 100º C. CAH treated with sugars and methylglyoxal showed a brown color with fructosamine and advanced glycation end-product formation. In all CAH-MRPs, the amyloid and carbonyl groups increased whereas the amino groups were reduced. Furthermore, CAH-MRPs at 100℃ for 8 hours had accelerated glycation with improved emulsifying activity, surface hydrophobicity, and antioxidant potential. FE-SEM FTIR, CD, NMR, and HPLC, analysis confirmed the structural changes with increased β-sheet and β-turn contents in CAH-MRPs. Differential scanning calorimetry indicated shifts in peak temperatures and endothermic transitions of enthalpy with better thermal stability. Overall results indicated that MRPs exhibited improved thermal, antioxidant, and emulsifying properties.
... Likewise, previous research has indicated that ligand binding can affect the conformational sampling by stabilizing functional alternate states of protein. Perticaroli et al. performed the dynamics study on an intrinsically disordered protein b-casein (CAS), where they applied neutron and light scattering techniques and found the CAS stiffens and partially folds upon calcium binding [69]. Döring et al. applied time-resolved tryptophan fluorescence anisotropy to maltose-binding protein and found that although the apo protein already has the alternate conformation which favors the binding to the ligand, the maltose-binding protein exhibits strong internal fluctuation that almost vanish upon ligand binding. ...
In this minireview we discuss the role of the more subtle conformational change—protein conformational sampling and connect it to the classic relationship of protein structure and function. The theory of pre-existing functional states of protein are discussed in context of alternate protein conformational sampling. Last, we discuss how temperature, ligand binding and mutations affect the protein conformational sampling mode which is linked to the protein function regulation. The review includes several protein systems that showed temperature dependent protein conformational sampling. We also specifically included two enzyme systems, thermophilic alcohol dehydrogenase (ht-ADH) and thermolysin which we previously studied when discussing temperature dependent protein conformational sampling.
... These belong to a class of 'intrinsically disordered proteins' (IDP) that lack stable three-dimensional structure (and whose structures are unknown). 29,30 Caseins transport calcium and can assemble as casein micelles which could potentially have conformational or linear epitopes or both. 31 Traditional thinking about conformational and linear epitopes may not apply to caseins or other IDP's. ...
Background
Patients are commonly challenged with foods containing baked milk, for example muffins, yet little is known about the specific allergen content of muffins used in milk challenges or of the effect that baking has on allergenicity.
Objective
Our objective was to compare the levels of major milk allergens in uncooked and baked muffins using monoclonal immunoassays and IgE antibody binding before and after baking.
Methods
Uncooked and baked muffins were prepared using recipes from Mount Sinai and Imperial College. Allergen levels were compared by ELISA for Bos d 5 (β‐lactoglobulin) and Bos d 11 (β‐casein). IgE reactivity was assessed using sera from milk‐sensitized donors in direct binding and inhibition ELISA.
Results
Bos d 5 was reduced from 680 µg/g in uncooked muffin mix to 0.17 µg/g in baked muffins, representing a >99% decrease after baking. Conversely, Bos d 11 levels in baked muffin remained high and only decreased by 30% from a mean of 4249 µg/g in uncooked muffin mix to 2961 µg/g when baked (~181 mg Bos d 11 per muffin). Baked muffins retained ~70% of the IgE binding to uncooked muffin mix. Baked muffin extract inhibited IgE binding to uncooked muffin mix by up to 80%, demonstrating retention of in vitro IgE reactivity.
Conclusions and Clinical Relevance
High levels of Bos d 11 in baked muffins pose a risk for adverse reactions, especially in patients who have high anti‐casein IgE antibodies.
... We (Permyakov et al., 2008). The structural flexibility of IDPs allows a broad functional repertoire 15 and a number of interaction partners (Uversky, 2009) to act and to influence protein function in 16 different processes, such as transcriptional regulation, translation, cellular signal transduction, 17 and storage of small molecules (Perticaroli et al., 2014). 18 Alongside with its disorder, Grh1 also shows an unexpected feature. ...
GRASPs are proteins involved in cell processes that seem paradoxical, such as being responsible for shaping the Golgi cisternae and also involved in unconventional secretion mechanisms that bypass the Golgi, among other functions in the cell. Despite its involvement in several relevant cell processes, there is still a considerable lack of studies on full-length GRASPs. Our group has previously reported an unexpected behavior of the full-length GRASP from the fungus C. neoformans : its intrinsically-disordered characteristic. Here, we generalize this finding by showing that is also observed in the GRASP from the yeast S. cerevisae (Grh1), which strongly suggests it may be a general property within the GRASP family. Furthermore, Grh1 is also able to form amyloid fibrils either upon heating or when submitted to changes in the dielectric constant of its surroundings, a condition that is experienced by the protein when in close contact with membranes of cell compartments, such as the Golgi apparatus. Intrinsic disorder and amyloid fibril formation can thus be two structural properties exploited by GRASP during its functional cycle.
... Low frequency collective vibrational dynamics in amorphous materials give rise to the Boson peak, the position of which scales with local rigidity in structural motifs so that IDPs, for example, are softer than b-sheet proteins which are softer than a-helical proteins (Perticaroli, Nickels, Ehlers, & Sokolov, 2014). Caseins have been the subject of a number of studies to show that rigidity is increased by calcium binding and local diffusive motions can be larger in amplitude than in globular proteins (Gaspar, Appavou, Busch, Unruh, & Doster, 2008;Perticaroli, Nickels, Ehlers, Mamontov, & Sokoov, 2014). A similar increase in rigidity of the phosphoprotein and IDP, osteopontin, was noted when it was complexed to CaP nanoclusters (Lenton, Seydel, et al., 2015). ...
Caseins are a group of closely related intrinsically disordered proteins (IDPs), best known for their occurrence in milk as stable, polydisperse, roughly spherical, amorphous particles, typically containing thousands of protein chains and hundreds of nanoclusters of calcium phosphate. The particles are called casein micelles though their structure bears no resemblance to detergent micelles. Caseins have an open and flexible conformation with a preponderance of poly-L-proline II secondary structure and hence cannot be described as hydrophobic proteins. Individually, and in combination, they associate through polar and non-polar interactions to form polydisperse fuzzy complexes (including the native casein micelle) while retaining their hydrated and flexible conformation to a large degree. Like many other IDPs, caseins are prone to form cytotoxic amyloid fibrils. However, they are also highly effective molecular chaperones so that a mixture of different caseins can form fuzzy complexes that are often self-limiting in size and, within which, amyloid fibril formation is suppressed. The remarkable ability of caseins to sequester nanoclusters of calcium phosphate in stable complexes is due to their flexible conformation and multiply-phosphorylated short sequences. These features combine to form a dense peptide shell around the calcium phosphate making the core-shell complex thermodynamically stable, even at high calcium and phosphate concentrations. Thus, the casein micelle provides a readily digested, high calcium food for the neonate. It also preserves the functional properties of caseins as IDPs and protects the mammary gland against amyloid formation and pathological calcification; dysfunctional processes that would reduce the future reproductive success of the mother.
... The radii of the D 2 O hydrated samples are close to those of folded globular proteins such as lysozyme, myoglobin or GFP (Pérez et al., 1999;Roh et al., 2006;Nickels et al., 2012). For the H 2 O hydrated samples, the radii are similar to those of b-casein, tRNA and E.coli (Dhindsa et al., 2014;Perticaroli et al., 2014;Roh et al., 2009;Jasnin et al., 2008). The difference in the diffusion radii of H 2 O and D 2 O hydrated samples may be due to either the different incoherent cross sections of hydrogen and deuterium, or to the solvent effect. ...
We present an investigation of the dynamics of calcium caseinate as a function of hydration, solvent isotope (H2O and D2O) and drying methods (roller drying and spray drying), using quasi-elastic neutron scattering (QENS). These factors are key to the formation of fibres in this material which makes it a potential candidate as a next-generation meat analogue. Using a phenomenological model, we find that the relaxation times of the dry spray dried powder decrease with increasing temperatures, while they do not change for the roller dried powder. The spectra of the hydrated samples reveal two independent picosecond processes, both reflecting localized re-orientational motions. We hypothesize that the faster motion is due to the external protein groups that are hydrophilic and the slower motion is due to the internal groups that are hydrophobic. The solvent effect of D2O is not limited to the external groups but prevails to the internal groups where less protons are mobile compared to the H2O hydrated samples. Higher temperatures narrow the number difference in mobile protons, possibly by altering the weak interactions inside the protein aggregates. These findings suggest that a harsh and longer drying process contributes to less active protein side-groups and highlight the hydrophobic effect of D2O on the fibre formation in calcium caseinate.
... Our results highlight a good stability of caseins to heat treatments, as already reported in literature [7]. The heat resistance of the caseins group seems ascribable to a well-defined disordered mobile structure (rheomorphic) and to the lack of co-operative transition of unfolding, or partial folding, during heating [29]. Indeed, caseins lack a rigid tertiary structure, which confers stability and develop a "random coil" conformation stabilized by hydrophobic interactions [30]. ...
Cow’s milk is considered the best wholesome supplement for children since it is highly enriched with micro and macro nutrients. Although the protein fraction is composed of more than 25 proteins, only a few of them are capable of triggering allergic reactions in sensitive consumers. The balance in protein composition plays an important role in the sensitization capacity of cow’s milk, and its modification can increase the immunological response in allergic patients. In particular, the heating treatments in the presence of a food matrix have demonstrated a decrease in the milk allergenicity and this has also proved to play a pivotal role in developing tolerance towards milk. In this paper we investigated the effect of thermal treatment like baking of cow’s milk proteins that were employed as ingredients in the preparation of muffins. A proteomic workflow was applied to the analysis of the protein bands highlighted along the SDS gel followed by western blot analyses with sera of milk allergic children in order to have deeper information on the impact of the heating on the epitopes and consequent IgE recognition. Our results show that incorporating milk in muffins might promote the formation of complex milk–food components and induce a modulation of the immunoreactivity towards milk allergens compared to milk baked in the oven at 180 °C for ten minutes. The interactions between milk proteins and food components during heating proved to play a role in the potential reduction of allergenicity as assessed by in vitro tests. This would help, in perspective, in designing strategies for improving milk tolerance in young patients affected from severe milk allergies.
... Neutron backscattering experiments have already explored protein solutions in a wide range of protein concentrations from below 50 mg/ml up to above 500 mg/ml [12,19]. Current topics of interest include for instance the investigation of protein cluster formation [11,15,17,23,24,25,26,27,28], the dynamics of intrinsically disordered proteins [8,29,30,31,32], the effect of crowding under pressure [33,34], and thermal unfolding [4,5,35,36,37]. ...
Novel cold neutron backscattering spectrometers contribute substantially to the understanding of the diffusive dynamics of proteins in dense aqueous suspensions. Such suspensions are fundamentally interesting for instance in terms of the so-called macromolecular crowding, protein cluster formation, gelation, and self-assembly. Notably, backscattering spectrometers with the highest flux can simultaneously access the center-of-mass diffusion of the proteins and the superimposed internal molecular diffusive motions. The nearly complete absence of protein-protein collisions on the accessible nanosecond observation time scale even in dense protein suspensions implies that neutron backscattering accesses the so-called short-time limit for the center-of-mass diffusion. This limit is particularly interesting in terms of a theoretical understanding by concepts from colloid physics. Here we briefly review recent progress in studying protein dynamics achieved with the latest generation of backscattering spectrometers. We illustrate this progress by the first data from a protein solution using the backscattering-and-time-of-flight option BATS on IN16B at the ILL and we outline future perspectives.
... During heat treatment of milk, β-Lg may be linked with κ-CN and α S2 -CN via thiolÀdisulfide exchange reactions, thereby increasing heat stability and inhibiting the precipitation of heat-denatured β-Lg (Singh, 1991). Unlike the intrinsic disordered structure of β-CN and α s1 -CN due to numerous proline residues and no disulfide bonds (Creamer, Richardson, & Parry, 1981;Perticaroli, Nickels, Ehlers, Mamontov, & Sokolov, 2014), the rigid globular structure of α-La and β-Lg could make them more resistant to proteolysis (Qin et al., 1998;Takagi, Teshima, Okunuki, & Sawada, 2003). In summary, knowledge of whey protein denaturation/aggregation processes is extremely useful for minimizing potential negative practical consequences and for improving functional properties of whey protein ingredients in many food applications. ...
... As before, we reduce the charge on nESI generated protein ions and examine the CCSDs of precursor and product ions. We compare the structural change when the charge is reduced for a rigid (BPTI) [48,49] and for an intrinsically disordered protein (beta casein) [50][51][52]. ...
Charge reduction in the gas phase provides a direct means of manipulating protein charge state, and when coupled to ion mobility mass spectrometry (IM-MS), it is possible to monitor the effect of charge on protein conformation in the absence of solution. Use of the electron transfer reagent 1,3-dicyanobenzene, coupled with IM-MS, allows us to monitor the effect of charge reduction on the conformation of two proteins deliberately chosen from opposite sides of the order to disorder continuum: bovine pancreatic trypsin inhibitor (BPTI) and beta casein. The ordered BPTI presents compact conformers for each of three charge states accompanied by narrow collision cross-section distributions (TWCCSDN2→He). Upon reduction of BPTI, irrespective of precursor charge state, the TWCCSN2→He decreases to a similar distribution as found for the nESI generated ion of identical charge. The behavior of beta casein upon charge reduction is more complex. It presents over a wide charge state range (9–28), and intermediate charge states (13–18) have broad TWCCSDN2→He with multiple conformations, where both compaction and rearrangement are seen. Further, we see that the TWCCSDN2→He of the latter charge states are even affected by the presence of radical anions. Overall, we conclude that the flexible nature of some proteins result in broad conformational distributions comprised of many families, even for single charge states, and the barrier between different states can be easily overcome by an alteration of the net charge.
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... 16 Since the BSA powder studied here is supposed to be dry, the QES most probably arises from the picosecond relaxational motions of side-chain residues in a cage formed by other residues or the protein backbone. 50 The quantity of residual water in our sample is too small to form the first hydration layer. So, the water molecules are most probably bound to the protein and may scarcely accomplish the reorientation motions observable in the 5−70 cm −1 region. ...
The present study focuses on protein motions on the picosecond time scale, generally characterized by the overlapping of vibrational and relaxational dynamics in disordered molecular systems. Recently it has been demonstrated that a dry protein, bovine serum albumin (BSA), shows a glass-like transition located in the temperature range between 240 K and 260 K. Here, we present the results of combined low-frequency Raman and inelastic neutron scattering studies of dry BSA at conditions close to this glass-like transition. The use of both techniques allows us to perform a detailed comparison of the dynamic susceptibility and the vibrational density of states of BSA obtained at different temperatures and to calculate the light-vibration coupling coefficient C(ω). Moreover, we analyzed the temperature evolution of the boson peak and a peak located at ~80cm(-1), which has previously been identified to originate from protein dynamics. We observe that both modes show an anomalous temperature behavior in the vicinity of Tg.
... While the majority of experiments conducted at CNCS are hard condensed matter studies, quasielastic scattering studies in soft matter materials make up a non-negligible part of the science program at CNCS. In the area of polymer and protein dynamics, various studies focused on the role of hydration water, [72,73] the nature of the collective vibrations ("boson peak"), [74,75] and the role of the secondary structure for the rigidity and functionality of these molecules [76][77][78]. These experiments are generally not intensity limited, unless the samples are fully deuterated. ...
The first eight years of operation of the Cold Neutron Chopper Spectrometer (CNCS) at the Spallation Neutron Source in Oak Ridge is being reviewed. The instrument has been part of the facility user program since 2009, and more than 250 individual user experiments have been performed to date. CNCS is an extremely powerful and versatile instrument and offers leading edge performance in terms of beam intensity, energy resolution, and flexibility to trade one for another. Experiments are being routinely performed with the sample at extreme conditions: T~0.05K, p>=2GPa and B=8T can be achieved individually or in combination. In particular, CNCS is in a position to advance the state of the art with inelastic neutron scattering under pressure, and some of the recent accomplishments in this area will be presented in more detail.
... This observation implies that the side chains of αSyn are surrounded by water molecules even in the fibril state. The dynamics of intrinsically disordered proteins, such as the tau protein and the casein protein, has been explored using neutron scattering, and the factors that affect the dynamics have been investigated [39,48,49]. In particular, the dynamics of hydration water was found to couple with the dynamics of the intrinsically disordered proteins [48]. ...
α-synuclein (αSyn) is a protein consisting of 140 amino acid residues and is abundant in the presynaptic nerve terminals in the brain. Although its precise function is unknown, the filamentous aggregates (amyloid fibrils) of αSyn have been shown to be involved in the pathogenesis of Parkinson's disease, which is a progressive neurodegenerative disorder. To understand the pathogenesis mechanism of this disease, the mechanism of the amyloid fibril formation of αSyn must be elucidated. Purified αSyn from bacterial expression is monomeric but intrinsically disordered in solution and forms amyloid fibrils under various conditions. As a first step toward elucidating the mechanism of the fibril formation of αSyn, we investigated dynamical behavior of the purified αSyn in the monomeric state and the fibril state using quasielastic neutron scattering (QENS). We prepared the solution sample of 9.5 mg/ml purified αSyn, and that of 46 mg/ml αSyn in the fibril state, both at pD 7.4 in D2O. The QENS experiments on these samples were performed using the near-backscattering spectrometer, BL02 (DNA), at the Materials and Life Science Facility at the Japan Accelerator Research Complex, Japan. Analysis of the QENS spectra obtained shows that diffusive global motions are observed in the monomeric state but largely suppressed in the fibril state. However, the amplitude of the side chain motion is shown to be larger in the fibril state than in the monomeric state. This implies that significant solvent space exists within the fibrils, which is attributed to the αSyn molecules within the fibrils having a distribution of conformations. The larger amplitude of the side chain motion in the fibril state than in the monomeric state implies that the fibril state is entropically favorable.
... The side chain dynamics depend on the length and polarity of the amino acid residue, as well as on the chemical environment of the side chain, which in turn is determined by the folding of the entire protein chain. Accordingly, an unfolded polypeptide chain is more flexible than a tightly folded globular protein [50,51]. ...
Myelin protein P2 is a fatty acid-binding structural component of the myelin sheath in the peripheral nervous system, and its function is related to its membrane binding capacity. Here, the link between P2 protein dynamics and structure and function was studied using elastic incoherent neutron scattering (EINS). The P38G mutation, at the hinge between the β barrel and the α-helical lid, increased the lipid stacking capacity of human P2 in vitro, and the mutated protein was also functional in cultured cells. The P38G mutation did not change the overall structure of the protein. For a deeper insight into P2 structure-function relationships, information on protein dynamics in the 10 ps to 1 ns time scale was obtained using EINS. Values of mean square displacements mainly from protein H atoms were extracted for wild-type P2 and the P38G mutant and compared. Our results show that at physiological temperatures , the P38G mutant is more dynamic than the wild-type P2 protein, especially on a slow 1-ns time scale. Molecular dynamics simulations confirmed the enhanced dynamics of the mutant variant, especially within the portal region in the presence of bound fatty acid. The increased softness of the hinge mutant of human myelin P2 protein is likely related to an enhanced flexibility of the portal region of this fatty acid-binding protein, as well as to its interactions with the lipid bilayer surface requiring conformational adaptations.
Thermal fluctuations of proteins at the ps-ns timescales are important for their functions and have extensively been studied using quasi-elastic neutron scattering (QENS). In general, the QENS spectra of proteins are analyzed in terms of two populations of atoms, i.e., the immobile fraction of atoms, the motions of which are too slow to be resolved within the instrumental energy resolution employed, and the mobile fraction of atoms, from which the average amplitude and frequency of protein atomic motions are characterized. On the other hand, it has been shown using molecular dynamics simulations that atomic motions are gradually enhanced as going from the protein core to the protein surface. Therefore, it is essential to further deconvolute the mobile fraction of atoms to study in detail the dynamical behavior of proteins. Here, an improved analytical model using QENS to deconvolute the mobile fraction of atoms into two populations of atoms, i.e., atoms with high mobility (HM atoms) and those with low mobility (LM atoms), is proposed. It was found that both the HM atoms and the LM atoms showed gradually enhanced dynamics with an increase in temperature even though any temperature-dependent terms are not included in the model. The presented model yields physically reasonable values for dynamical parameters and hence its future application will be useful to understand the molecular mechanism of various protein functions where atoms with higher mobility on or close to the protein surface play a crucial role.
Intrinsically disordered proteins are ubiquitous throughout all known proteomes, playing essential roles in all aspects of cellular and extracellular biochemistry. To understand their function, it is necessary to determine their structural and dynamic behavior and to describe the physical chemistry of their interaction trajectories. Nuclear magnetic resonance is perfectly adapted to this task, providing ensemble averaged structural and dynamic parameters that report on each assigned resonance in the molecule, unveiling otherwise inaccessible insight into the reaction kinetics and thermodynamics that are essential for function. In this review, we describe recent applications of NMR-based approaches to understanding the conformational energy landscape, the nature and time scales of local and long-range dynamics and how they depend on the environment, even in the cell. Finally, we illustrate the ability of NMR to uncover the mechanistic basis of functional disordered molecular assemblies that are important for human health.
Intrinsically disordered proteins (IDPs) are proteins that, in comparison with globular/structured proteins, lack a distinct tertiary structure. Here, we use the model IDP, Histatin 5, for studying its dynamical properties under self-crowding conditions with quasi-elastic neutron scattering in combination with full atomistic molecular dynamics (MD) simulations. The aim is to determine the effects of crowding on the center-of-mass diffusion as well as the internal diffusive behavior. The diffusion was found to decrease significantly, which we hypothesize can be attributed to some degree of aggregation at higher protein concentrations, (≥100 mg/mL), as indicated by recent small-angle X-ray scattering studies. Temperature effects are also considered and found to, largely, follow Stokes-Einstein behavior. Simple geometric considerations fail to accurately predict the rates of diffusion, while simulations show semiquantitative agreement with experiments, dependent on assumptions of the ratio between translational and rotational diffusion. A scaling law that previously was found to successfully describe the behavior of globular proteins was found to be inadequate for the IDP, Histatin 5. Analysis of the MD simulations show that the width of the distribution with respect to diffusion is not a simplistic mirroring of the distribution of radius of gyration, hence, displaying the particular features of IDPs that need to be accounted for.
The influence of ultrasound treatment on the subsequent glycation process of proteins is controversial. Glycation behaviors of bovine serum albumin (BSA), β-lactoglobulin (β-Lg) and β-casein (β-CN) after ultrasound pretreatment (UP) were compared by both evaluating glycation kinetics and analyzing structural changes of proteins. UP resulted in both unfolding and aggregation behavior in protein samples, which altered the accessibility of the Lys and Arg. Five cycles of UP up-regulated the glycation degree of BSA and β-Lg, possibly due to the unfolding behavior induced by UP, which exposed additional glycation sites. In contrast, 30 cycles of UP induced a dramatic increase (by 97.9 nm) in particle size of BSA, thus burying portions of glycation sites and suppressing the glycation process. Notably, UP had minimal influence on glycation kinetics of β-CN, due to its intrinsic disordered structure. Based on proteomics analysis, the preference of Lys and Arg during glycation was found to be changed by UP in BSA and β-Lg. Four, 3 and 3 unique carboxyethylated lysine residues were identified in glycated BSA after 0, 5 and 30 cycles of UP, respectively. This study suggests that the protein glycation can be affected by UP, depending on the ultrasonication duration and native structure of the protein.
The dynamics of proteins in solution includes a variety of processes, such as backbone and side-chain fluctuations, interdomain motions, as well as global rotational and translational (i.e. center of mass) diffusion. Since protein dynamics is related to protein function and essential transport processes, a detailed mechanistic understanding and monitoring of protein dynamics in solution is highly desirable. The hierarchical character of protein dynamics requires experimental tools addressing a broad range of time-and length scales. We discuss how different techniques contribute to a comprehensive picture of protein dynamics, and focus in particular on results from neutron spectroscopy. We outline the underlying principles and review available instrumentation as well as related analysis frameworks.
Polysorbates are widely used in food and cosmetic products and pharmaceuticals for emulsion stabilization, preventing surface absorption and as stabilizers against protein aggregation. But their impacts on the secondary structure and conformational stabilization of bovine casein, a typical intrinsically disordered protein (IDP), have not been fully addressed so far, which is crucial to evaluate the performance of polysorbates as stabilizer for food products containing bovine casein. Here, we assessed the effects of polysorbate 20 (PS-20)and polysorbate 80 (PS-80)on the secondary structure and conformation of bovine casein micelles by fluorescence and circular dichroism spectra, dynamic light scattering (DLS), small angle x-ray scattering (SAXS)and transition electron microscopy (TEM). In addition, three-dimensional structure of bovine β-casein, a major component of bovine casein, was predicted using homology modelling with the aid of PSIPRED server to outline the potential binding sites of bovine casein for polysorbates. We found that PS-20 presented a higher binding affinity (K a = 3.76 × 10 ⁶ M ⁻¹ )and stoichiometry (n = 0.89)to bovine casein than that of PS-80 (K a = 1.45 × 10 ⁶ M ⁻¹ , n = 0.45). The α-helical and β-sheet conformation of bovine casein was increased upon binding with PS-20 whereas binding of PS-80 had little impact on secondary structural elements of this milk protein. Moreover, bovine casein micelles were dissociated by PS-20, which co-assembled into micellar-like particles with a rather spherical shape and homogenous size distribution ranging from 20 to 50 nm in diameter. The lauric acid chains may bind with disordered regions connected by colloidal calcium phosphate (CCP)as the core, and the sorbitan groups may associate with N-terminal outwards as the shell providing hydrophilicity to stabilize the polysorbates-casein complex. On the contrary, PS-80 led to formation of sponge-like aggregates with the radius ranging from 200 to 500 nm as indicated by the data of DLS, SAXS and TME. We assume that the stearic hindrance originating from oleic group and the kink due to the cis-double bond in the sidechain of PS-80 contribute to the aggregation of bovine casein induced by PS-80. Furthermore, a tentative binding mechanism of polysorbates with bovine casein micelles and model of complexes were proposed to illustrate the polysorbates-induced structural change and conformational stabilization of bovine casein micelles. Thus, we argue that the difference in binding of polysorbates to bovine casein may influence the functional properties (emulsification, foaming ability, etc)of milk protein. Taken together, we suggest that PS-20 may be preferred option as potential protein stabilizer for dairy products, and careful consideration should be given when selecting PS-80 as stabilizer for bovine casein, a typical IDP.
A predicted three-dimensional structure of bovine β-casein was constructed using homology modeling with the aid of MODELLER and I-TASSER programs, with the validity and reliability of the models evaluated according to stereochemical qualities and small angle X-ray scattering. By comparing results from the two models using the CRYSOL program, an optimal model of β-casein structure derived from I-TASSER, was selected and used in subsequent molecular dynamics (MD) analysis. 300 ns MD simulations of β-casein in water and with the presence of different SDS concentrations at 300 K were performed. The results of the MD simulations indicated that SDS molecules played a dual role in modifying the conformation of β-casein at 300 K. SDS concentrations below its CMC (1 mM), at which only the monomer form of SDS was present, induced β-casein to lose its secondary structure by converting helices into random coils; however the conformation of the complex was still comparable with that of native β-casein. In the presence of 10 mM SDS (above its CMC), the helical content of β-casein was increased along with reduced random coil, and the structural rearrangement led to a more compact conformation. The latter change is likely related to the hydrophobic interactions that dominate the binding of the C-terminal region, along with the anchoring of sulfate groups of SDS on the positively charged N-terminal portion via electrostatic attraction. Hydrogen bonding supplemented the SDS-induced stabilization of β-casein. A correlated “necklace and bead” model, in which the micelles nucleate on the protein hydrophobic sites, was proposed for the structure of β-casein-SDS complexes.
GRASPs are proteins involved in cell processes that seem paradoxical: responsible for shaping the Golgi cisternae and involved in unconventional secretion mechanisms that bypass the Golgi. Despite its physiological relevance, there is still a considerable lack of studies on full-length GRASPs. Our group has previously reported an unexpected behavior of the full-length GRASP from the fungus C. neoformans: its intrinsically-disordered characteristic. Here, we generalize this finding by showing that it is also observed in the GRASP from S. cerevisae (Grh1), which strongly suggests it might be a general property within the GRASP family. Furthermore, Grh1 is also able to form amyloid-like fibrils either upon heating or when submitted to changes in the dielectric constant of its surroundings, a condition that is experienced by the protein when in close contact with membranes of cell compartments, such as the Golgi apparatus. Intrinsic disorder and fibril formation can thus be two structural properties exploited by GRASP during its functional cycle.
With well-known nutritional properties, casein contributes to about 80% of protein content in milk and has been classified as highly intrinsically disordered protein (IDP). In this paper, the sulfate dodecyl sodium (SDS)-induced conformational changes of bovine casein were studied by multi-techniques. Isothermal titration calorimetry (ITC) and differential scanning calorimetry (DSC) were used to obtain the stoichiometry of conformational changes and the thermal stability of the formed complexes. Spectral results indicated that casein presented a higher helical content but loss of tertiary structure above critical micelle concentration of SDS, namely, the so-called molten globule like state. The thermal self-association of casein could be prevented by SDS according to far-UV CD even at 70 °C. The ¹H NMR spectrum of casein showed that the resonance around 1.0 ppm, the region of α-hydrogen, shifted to the higher field, and the aromatic region around 5.5–8.0 ppm shifted to the lower field, while the NOESY spectra of casein exhibited few chemical shifts with binding of SDS. Combining the results of dynamic light scattering (DLS), scanning electron microscope (SEM) and small angle x-ray scattering (SAXS), one obtains that casein micelles presented an elliptical shape of ∼800 nm in diameter and upon binding with SDS, the casein micelles disassociated into more compact globular particles of 10 nm in diameter with a core-shell structure composed by SDS molecules and casein proteins. The present work, not only provides molecular insights into the mechanism of SDS-induced stability of a model IDP, casein, but also helps understand the role of surfactants on the structure–function relationship of bovine casein in the food industry.
Using active lactose to hydrolyze lactose during storage is a common process to produce lactose-hydrolyzed (LH) milk. Proteolysis induced by residual proteases in commercial lactase was studied in a system using purified β-casein or β-lactoglobulin during a 60-day storage period at 22 °C or 38 °C. The proteolysis of β-casein by residual proteases occurred more extensively than that of β-lactoglobulin. Peptidomic analysis by LC-ESI-MS/MS revealed that Ile, Leu, Tyr and Phe residues near the C-terminus of β-casein were the main sites of cleavage by the residual proteases, generating assumed bitter peptides. In the subsequent in vitro digestion study, proteolysis during storage was shown to greatly affect the subsequent digestibility of β-casein, leading to an elevated degree of hydrolysis and the formation of new digested peptides. This study highlights the potential influence of residual proteases in commercial lactase on the storage quality and digestibility of LH milk containing active lactase.
Nuclear magnetic resonance (NMR) spectroscopy is one of the most powerful experimental approaches for investigating the conformational behavior of intrinsically disordered proteins (IDPs). IDPs represent a significant fraction of all proteomes, and, despite their importance for understanding fundamental biological processes, the molecular basis of their activity still remains largely unknown. The functional mechanisms exploited by IDPs in their interactions with other biomolecules are defined by their intrinsic dynamic modes and associated timescales, justifying the considerable interest over recent years in the development of technologies adapted to measure and describe this behavior. NMR spin relaxation delivers information-rich, site-specific data reporting on conformational fluctuations occurring throughout the molecule. Here we review recent progress in the use of ¹⁵N relaxation to identify local backbone dynamics and long-range chain-like motions in unfolded proteins.
In this article, we elucidate the protein activity from the perspective of protein softness and flexibility by studying the collective phonon-like excitations in a globular protein, human serum albumin (HSA) and taking advantage of the state-of-the-art inelastic X-ray scattering (IXS) technique. Such excitations demonstrate that the protein becomes softer upon thermal denaturation due to disruption of weak non-covalent bonds. On the other hand, no significant change in the local excitations is detected in ligand- (drugs) bound HSA compared to the ligand-free HSA. Our results clearly suggest that the protein conformational flexibility and rigidity are balanced by the native protein structure for biological activity.
This contribution highlights the recently developed microscopic picture of the effects of hydration and electrostatic interactions on subnanosecond dynamics of biopolymers protein and ribonucleic acid (RNA), studied by quasielastic neutron scattering spectroscopy. In contrast to the traditional concept of water-slaved dynamics, more detailed analysis of the dynamics of different chemical structures (lysozyme vs transfer RNA; electrostatically unscreened vs screened) demonstrates that chemical and physical responses of biopolymers to hydration and charge screening determine the dynamic interactions. How the relationship of the dynamical flexibility and structural stability varies depending on water-driven or charge screening-driven folding into biologically active structures has also been discussed. However, the biological relevance of the fast conformational dynamics still remains elusive. Exploring the dynamic heterogeneity of biopolymers is proposed as a potential approach to the identification of biologically important dynamics.
Proteins that do not have a well-defined structure in their functional state are referred to as intrinsically disordered proteins (IDPs). IDPs are ubiquitous in biological cells and their aggregation is involved in many diseases. The extended conformations of IDPs result in a large water interface, yet, interactions between IDPs and water are only scarcely documented. Water has been termed the matrix of life because it is essential for a variety of molecular processes, including protein folding, stability, and activity. The IDP tau regulates microtubule activity in neurons and is known to form amyloid fibers that are one of the hallmarks of Alzheimer disease. In this PhD thesis, the biological relevance of water dynamics around IDPs is addressed. We combine computational and experimental approaches, including all-atom MD simulations, incoherent neutron scattering, terahertz spectroscopy and small angle X-ray scattering, to study the hydration water dynamics of the tau protein in its native and fibrillated states. Firstly, a translational diffusion of hydration water molecules is found to be essential for biologically relevant dynamics of both IDPs and globular proteins. Secondly, compared to monomers, we find an enhancement of hydration water mobility around tau amyloid fibers that is suggested to play a role in fiber formation. Finally, the investigation of collective water dynamics reveals that the tau protein influences about two times less water molecules than a globular protein, which might be involved in tau's binding mechanisms. In conclusion, this piece of work investigated the dynamical properties of water around IDPs and suggests that the hydration water dynamics might play fundamental roles in binding and aggregation of IDPs.
The tau protein, whose aggregates are involved in Alzheimer's disease, is an intrinsically disordered protein (IDP) that regulates microtubule activity in neurons. An IDP lacks a single, well-defined structure and, rather, constantly exchanges among multiple conformations. In order to study IDP dynamics, the combination of experimental techniques, such as neutron scattering, and computational techniques, such as molecular dynamics (MD) simulations, is a powerful approach. Amorphous hydrated powder samples have been very useful for studying protein internal dynamics experimentally, e.g. using neutron scattering. Thus, there is demand for realistic in silico models of hydrated protein powders. Here we present an MD simulation analysis of a powder hydrated at 0.4 g water / g protein of the IDP tau in the temperature range 20-300 K. By comparing with neutron scattering data, we identify the protein-water interface as the predominant feature determining IDP dynamics. The so-called protein dynamical transition is shown to be attenuated, but not suppressed, in the parts of the protein that are not exposed to the solvent. In addition, we find similarities in the mean-squared displacements of the core of a globular protein and 'dry' clusters formed by the IDP in hydrated powders. Thus, the ps to ns dynamics of proteins in hydrated powders originate mainly from those residues in contact with solvent. We propose that by measuring the dynamics of protein assemblies, such as aggregates, one might assess qualitatively their state of hydration.
We present an overview of protein dynamics based mostly on results of neutron scattering, dielectric relaxation spectroscopy and molecular dynamics simulations. We identify several major classes of protein motions on the time scale from faster than picoseconds to several microseconds, and discuss the coupling of these processes to solvent dynamics. Our analysis suggests that the microsecond backbone relaxation process might be the main structural relaxation of the protein that defines its glass transition temperature, while faster processes present some localized secondary relaxations. Based on the overview, we formulate a general picture of protein dynamics and discuss the challenges in this field.
YY1 (Yin Yang 1) is a zinc finger protein with an essential role in various biological functions via DNA- and protein-protein interactions with numerous partners. YY1 is involved in the regulation of a broad spectrum of cellular processes such as embryogenesis, proliferation, tumorigenesis, and snRNA transcription. The more than 100 reported targets of the YY1 protein suggest that it contains intrinsically disordered regions that are involved in such diverse interactions. Here, we present a study of the structural properties of human YY1 using several biochemical and biophysical techniques (fluorescence, circular dichroism, gel filtration chromatography, proteolytic susceptibility) together with various bioinformatics approaches. To facilitate our exploration of the YY1 structure, the full-length protein as well as an N-terminal fragment (residues 1 - 295) and the C-terminal DNA binding domain were used. We found the N-terminus to be a non-compact fragment of YY1 with little residual secondary structure and lacking a well-defined tertiary structure. The results of our study indicate that YY1 belongs to the family of intrinsically disordered proteins (IDPs), which exist natively in a partially unfolded conformation. This article is protected by copyright. All rights reserved.
© 2015 Wiley Periodicals, Inc.
Protein low-frequency vibrational modes are an important portion of a proteins' dynamical repertoire. Yet, it is notoriously difficult to isolate specific vibrational features in the spectra of proteins. Given an appropriately chosen model peptide, and using different experimental conditions, we can simplify the system and gain useful insights into the protein vibrational properties. Combining neutron scattering, depolarized light scattering, and molecular dynamics simulations, we analyse the low frequency vibrations of biological molecules, comparing the results from a small globular protein, lysozyme, and an amphiphilic peptide, NALMA, both in solution and in powder states. Lysozyme and NALMA present similar spectral features in the frequency range between 1 and 10 THz. With the aid of MD simulations, we assign the spectral features to methyl groups' librations (1–5 THz) and hindered torsions (5–10 THz) in NALMA. Our data also show that, while proteins display boson peak vibrations in both powder and solution forms, NALMA exhibits boson peak vibrations in powder form only. This provides insight into the nature of this feature, suggesting a connection of BP collective motions to a characteristic length scale of heterogeneities present in the system. These results provide context for the use of model peptide systems to study protein dynamics; demonstrating both their utility, and the great care that has to be used in extrapolating results observed in powder to solutions.
Defining precisely the mechanical properties of bio-macromolecular systems on a nanometer length scale is crucial for the development of more efficient drug delivery systems and scaffolds-based tissue engineering. We characterized the structure, topology, and rigidity properties of poly-L-glutamic acid (PGA), prepared with different molecular weights and secondary structures, using the same approach that we recently applied to proteins1-3. We employed various techniques, including FT-IR, SEM, light scattering, neutron diffraction, and neutron scattering spectroscopy.
The dynamics of proteins in solution is a complex and hierarchical process, affected by the aqueous environment as well as temperature. We present a comprehensive study on nanosecond time and nanometer length scales below, at, and above the denaturation temperature $T_d$. Our experimental data evidence dynamical processes in protein solutions on three distinct time scales. We suggest a consistent physical picture of hierarchical protein dynamics: (i) Self-diffusion of the entire protein molecule is confirmed to agree with colloid theory for all temperatures where the protein is in its native conformational state. At higher temperatures $T>T_d$, the self-diffusion is strongly obstructed by cross-linking or entanglement. (ii) The amplitude of backbone fluctuations grows with increasing $T$, and a transition in its dynamics is observed above $T_d$. (iii) The number of mobile side-chains increases sharply at $T_d$ while their average dynamics exhibits only little variations. The combination of quasi-elastic neutron scattering and the presented analytical framework provides a detailed microscopic picture of the protein molecular dynamics in solution, thereby reflecting the changes of macroscopic properties such as cluster formation and gelation.
Natively unfolded (intrinsically disordered) proteins have attracted growing attention due to their high abundance in nature, involvement in various signalling and regulatory pathways and direct association with many diseases. In the present work the combined effect of temperature and alcohols, trifluoroethanol (TFE) and hexafluoroisopropanol (HFIP), on the natively unfolded 4E-BP1 protein was studied to elucidate the balance between temperature-induced folding and unfolding in intrinsically disordered proteins. It was shown that elevated temperatures induce reversible partial folding of 4E-BP1 both in buffer and in the mixed solutions containing denaturants. In the mixed solutions containing TFE (HFIP) 4E-BP1 adopts a partially folded helical conformation. As the temperature increases, the initial temperature-induced protein folding is replaced by irreversible unfolding/melting only after a certain level of the protein helicity has been reached. Onset unfolding temperature decreases with TFE (HFIP) concentration in solution. It was shown that an increase in the temperature induces two divergent processes in a natively unfolded protein—hydrophobicity-driven folding and unfolding. Balance between these two processes determines thermal behaviour of a protein. The correlation between heat-induced protein unfolding and the amount of helical content in a protein is revealed. Heat-induced secondary structure formation can be a valuable test to characterise minor changes in the conformations of natively unfolded proteins as a result of site-directed mutagenesis. Mutants with an increased propensity to fold into a structured form reveal different temperature behaviour.
There is tremendous interest in understanding the role that secondary structure plays in the rigidity and dynamics of proteins. In this work we analyze nanomechanical properties of proteins chosen to represent different secondary structures: [small alpha]-helices (myoglobin and bovine serum albumin), [small beta]-barrels (green fluorescent protein), and [small alpha] + [small beta] + loop structures (lysozyme). Our experimental results show that in these model proteins, the [small beta] motif is a stiffer structural unit than the [small alpha]-helix in both dry and hydrated states. This difference appears not only in the rigidity of the protein, but also in the amplitude of fast picosecond fluctuations. Moreover, we show that for these examples the secondary structure correlates with the temperature- and hydration-induced changes in the protein dynamics and rigidity. Analysis also suggests a connection between the length of the secondary structure ([small alpha]-helices) and the low-frequency vibration
Caseins are members of a class of proteins with extremely open and flexible conformations. Here, we consider what features of their sequences are important in maintaining such a structure. Primary structures of the αS1-, β- and κ-caseins from species including the cow, sheep, rat, mouse, rabbit and guinea pig were aligned both by a variety of automatic multiple alignment procedures, and manually, to identify conserved features. Fully conserved residues in the mature proteins were unusually rare and involved mainly residues that have high mutation rates in conventional alignment scoring schemes. Autocorrelation of sequences using residue mutation rate scores as measures of similarity revealed that around the PQNI conserved sequence of β-caseins there appear to be repeated sequences similar to Pro-rich domain-linking peptides found in a number of other proteins. Other cryptic repeats were found in αS1-caseins. Predicted secondary structures were calculated and it is argued that apart from the regions around the centres of phosphorylation, the caseins are essentially of the all-β-strand type. However, condensation into β-sheets is inhibited by certain of the conserved features of the primary structure, allowing the proteins to maintain an open and mobile (rheomorphic) conformation.
The secondary structure of caseins was investigated with resolution-enhanced laser Raman spectroscopy. Raman spectra in the 1580 to 1720 cm -1 region were obtained from the following lyophilized proteins: 1) asl-casein , 2) /3-casein, 3) a natural mixture of bovine whole casein, and 4) micelles of the natural mixture in the presence of Ca 2+ ions. In addition, /3-casein was also investigated in D20 solution. The spectra obtained were Fourier deconvolved and curve fitted with Gaussian components. The results suggest that both as1 and /3-casein have around 10% helical structure, around 20% /3-structure, and from 20 to 35% turns. The turns are clearly distinguishable from the moiety usually called undefined, random, or structureless. Freeze-dried micelles in the presence of Ca 2+ ions and submicelles in the presence of K + ions appear to contain an increased amount of turns and of /3-structure as compared with the as1- and 3-caseins. The increase in turns is at the expense of the amount of undefined structure. All conformational designations here are based on spectroscopic assignments de- rived from crystallized proteins with well characterized structures. These designa- tions thus have a more qualitative, descriptive meaning for caseins than for other milk proteins, such as a-lactalbumin or 3-1actoglobulin.
Recent studies have discovered strong differences between the dynamics of nucleic acids (RNA and DNA) and proteins, especially at low hydration and low temperatures. This difference is caused primarily by dynamics of methyl groups that are abundant in proteins, but are absent or very rare in RNA and DNA. In this paper, we present a hypothesis regarding the role of methyl groups as intrinsic plasticizers in proteins and their evolutionary selection to facilitate protein dynamics and activity. We demonstrate the profound effect methyl groups have on protein dynamics relative to nucleic acid dynamics, and note the apparent correlation of methyl group content in protein classes and their need for molecular flexibility. Moreover, we note the fastest methyl groups of some enzymes appear around dynamical centers such as hinges or active sites. Methyl groups are also of tremendous importance from a hydrophobicity/folding/entropy perspective. These significant roles, however, complement our hypothesis rather than preclude the recognition of methyl groups in the dynamics and evolution of biomolecules.Electronic supplementary material The online version of this article (doi:10.1007/s10867-012-9268-6) contains supplementary material, which is available to authorized users.
Flexibility, or softness, is crucial for protein function and consists of a conformational component, involving jumps between potential wells, and an elastic component, involving fluctuations within the wells. Combining molecular dynamics simulation with incoherent neutron scattering and light scattering measurements on green fluorescent protein, we reveal a relationship between the intrawell fluctuations and elastic moduli of the protein. This finding leads to a simple means of experimentally separating the conformational from the elastic atomic displacements.
Expanded polyglutamine repeats have been proposed to cause neuronal degeneration in Huntington's disease (HD) and related
disorders, through abnormal interactions with other proteins containing short polyglutamine tracts such as the transcriptional
coactivator CREB binding protein, CBP. We found that CBP was depleted from its normal nuclear location and was present in
polyglutamine aggregates in HD cell culture models, HD transgenic mice, and human HD postmortem brain. Expanded polyglutamine
repeats specifically interfere with CBP-activated gene transcription, and overexpression of CBP rescued polyglutamine-induced
neuronal toxicity. Thus, polyglutamine-mediated interference with CBP-regulated gene transcription may constitute a genetic
gain of function, underlying the pathogenesis of polyglutamine disorders.
Neutron spectroscopy has been used to probe picosecond to nanosecond dynamics in lyophilised apoferritin, insulin, superoxide dismutase and green fluorescent protein. These proteins have markedly
different secondary structures yet similar CH3 compositions. Results suggest that while only CH3 activation is apparent in apoferritin, an enhanced dynamic environment presents itself in Ins, SOD and GfP. Our results hint at a structure dependent dynamic landscape.
In the development of new foods or in the control of traditional process, protein functionality plays a paramount role. It has long been theorized that changes in protein structure can alter functionality, but there has been a lack of reliable methodologies for observing protein structural changes in ‘real-world’ food samples. Here, a technique for determination of the global secondary structure of proteins using Fourier transform infrared (FTIR) spectroscopy in H2O instead of D2O is assessed and contrasted with other methodologies for structural determinations. A quantitative procedure is presented for preparing and analysing FTIR spectra of proteins for the determination of their global secondary structural components. As an example, an analysis of the FTIR spectra of egg-white lysozyme is presented and correlated with global secondary structure values calculated from its X-ray crystallographic structure. Other examples of FTIR analyses of food proteins are presented with consideration given to qualitative procedures for more rapid structural analysis of processed samples.
To obtain a molecular basis for the similarities and dissimilarities in the functional, chemical, and biochemical properties between β-casein and the other caseins, three-dimensional models have been presented. Secondary structural prediction algorithms and molecular modeling techniques were used to predict β-casein structure. The secondary structure of bovine β-casein was re-examined using Fourier transform infrared and circular dichroism spectroscopies to test these predictions. Both methods predict a range of secondary structures for β-casein (28–32% turns, 32–34% extended) at 25°C. These elements were highly stable from 5 to 70°C as viewed by circular dichroism. More flexible conformational elements, tentatively identified as loops, helix and short segments of polyproline II, were influenced by temperature, increasing with elevated temperatures. Another view is that as temperature decreases, these elements are lost (cold denaturation). Several distinct transitions were observed by circular dichroism at 10, 33 and 41°C, and another transition, extrapolated to occur at 78°C. Calculations from analytical ultracentrifugation indicate that the 10, 33 and 41°C transitions occur primarily in the monomeric form of the protein. As β-casein polymers are formed, and increase in size, the transitions at higher temperature may reflect changes in the more flexible conformational elements as they adjust to changes in surface charge during polymer formation. The transition at 10°C may represent an actual general conformational change or cold denaturation. Over the range of temperatures studied, the sheet and turn areas remain relatively constant, perhaps forming a supporting hydrophobic core for the monomers within the micelle-like polymer. This interpretation is in accord with the known properties of β-casein, and those predicted from molecular modeling.
A simplified description of the 295 K dynamics of a globular protein over a wide frequency range (1-1000 GHz) is obtained by combining neutron scattering of lysozyme with molecular dynamics simulation. The molecular dynamics simulation agrees quantitatively with experiment for both the protein and the hydration water and shows that, whereas the hydration water molecules subdiffuse, the protein atoms undergo confined motion decomposable into three distinct classes: localized diffusion, methyl group rotations, and jumps. Each of the three classes gives rise to a characteristic neutron susceptibility signal.
The design and performance of the new cold neutron chopper spectrometer (CNCS) at the Spallation Neutron Source in Oak Ridge are described. CNCS is a direct-geometry inelastic time-of-flight spectrometer, designed essentially to cover the same energy and momentum transfer ranges as IN5 at ILL, LET at ISIS, DCS at NIST, TOFTOF at FRM-II, AMATERAS at J-PARC, PHAROS at LANSCE, and NEAT at HZB, at similar energy resolution. Measured values of key figures such as neutron flux at sample position and energy resolution are compared between measurements and ray tracing Monte Carlo simulations, and good agreement (better than 20% of absolute numbers) has been achieved. The instrument performs very well in the cold and thermal neutron energy ranges, and promises to become a workhorse for the neutron scattering community for quasielastic and inelastic scattering experiments.
We use elastic neutron scattering to demonstrate that a sharp increase in the mean-squared atomic displacements, commonly observed in hydrated proteins above 200 K and often referred to as the dynamical transition, is present in the hydrated state of both native and denatured lysozyme. A direct comparison of the native and denatured protein thus confirms that the presence of the transition in the mean-squared atomic displacements is not specific to biologically functional molecules.
Structural fluctuations in proteins on the picosecond timescale have been studied in considerable detail by theoretical methods such as molecular dynamics simulation, but there exist very few experimental data with which to test the conclusions. We have used the technique of inelastic neutron scattering to investigate atomic motion in hydrated myoglobin over the temperature range 4-350 K and on the molecular dynamics timescale 0.1-100 ps. At temperatures below 180 K myoglobin behaves as a harmonic solid, with essentially only vibrational motion. Above 180 K there is a striking dynamic transition arising from the excitation of nonvibrational motion, which we interpret as corresponding to torsional jumps between states of different energy, with a mean energy asymmetry of 12 kJ mol-1. This extra mobility is reflected in a strong temperature dependence of the mean-square atomic displacements, a phenomenon previously observed specifically for the heme iron by Mössbauer spectroscopy, but on a much slower timescale (10(-7) s). It also correlates with a glass-like transition in the hydration shell of myoglobin and with the temperature-dependence of ligand-binding rates at the heme iron, as monitored by flash photolysis. In contrast, the crystal structure of myoglobin determined down to 80 K shows no significant structural transition. The dynamical behaviour we find for myoglobin (and other globular proteins) suggests a coupling of fast local motions to slower collective motions, which is a characteristic feature of other dense glass-forming systems.
In order to characterize the dynamic properties of the denatured state of staphylococcal nuclease, R1, R2, and NOE relaxation parameters have been measured for the backbone 15N nuclei of a 131 residue fragment that serves as a model of the denatured state under non-denaturing conditions. The relaxation data indicate a wide range of amplitudes for segmental motion and are inconsistent with a random coil conformation. An optimal value of 7.8 ns was obtained for the molecular rotational correlation time tau m based on the analysis of the 79 residues for which R1, R2, and NOE relaxation data could be obtained. This value corresponds roughly to the slowest detectable motion on the nanosecond time scale and is of a magnitude consistent with global tumbling of a large portion of the molecule. For the majority of residues, experimental data could be described most adequately in terms of a modified "model-free" formalism which includes contributions from internal motions on both an intermediate (tau e) and a fast time scale (tau f) in the context of slow overall tumbling (tau m). The generalized order parameters S2, which gives the amplitude of motions on time scales faster than tau m, correlates with sequence hydrophobicity and suggests a relationship between chain flexibility and sequence propensity for hydrophobic collapse. The fractional populations of three alpha-helices in the protein show a stronger correlation with S2 values and hydrophobicities than with intrinsic helix propensities. These observations suggest that secondary structure may be preferentially stabilized in hydrophobic segments of the sequence.
To obtain a molecular basis for the similarities and dissimilarities in the functional, chemical, and biochemical properties between beta-casein and the other caseins, a predicted three-dimensional model is presented. The predicted structure was assembled using molecular modeling techniques, as well as secondary structural prediction algorithms, in conjunction with global secondary structural information from Raman spectroscopy. To add validity to this model, the structure was refined using energy minimization techniques to diminish the likelihood of structural overlaps and energetically unfavorable van der Waals contacts arising from the large number of proline residues present in the beta-casein sequence. The refined model overall showed a loosely packed, asymmetrical structure with an axial ratio of 2:1. Hydrophobic side chains were uniformly dispersed over one end (C terminal) and the center surface of the structure; the other end (N terminal) was hydrophilic. The hydrophobic section also possessed a large loop through which water could easily travel. Such a suprasurfactant structure could account for the micellar type of hydrophobically driven self-association exhibited by beta-casein. Other chemical and biochemical data are in good agreement with the refined structure.
Quasielastic incoherent neutron scattering from hydrogen atoms, which are distributed nearly homogeneously in biological molecules, allows the investigation of diffusive motions occurring on the pico- to nanosecond time scale. A quasielastic incoherent neutron scattering study was performed on the integral membrane protein bacteriorhodopsin (BR), which is a light-driven proton pump in Halobacterium salinarium. BR is embedded in lipids, forming patches in the cell membrane of the organism, which are the so called purple membranes (PMs). Measurements were carried out at room temperature on oriented PM-stacks hydrated at two different levels (low hydration, h = 0.03 g of D2O per g of PM; high hydration, h = 0.28 g of D2O per g of PM) using time-of-flight spectrometers. From the measured spectra, different diffusive components were identified and analyzed with respect to the influence of hydration. This study supports the idea that a decrease in hydration results in an appreciable decrease in internal molecular flexibility of the protein structure. Because it is known from studies on the function of BR that the pump activity is reduced if the hydration level of the protein is insufficient, we conclude that the observed diffusive motions are essential for the function of this protein. A detailed analysis and classification of the different kinds of diffusive motions, predominantly occurring in PMs under physiological conditions, is presented.
Multidimensional NMR studies of proteins in unfolded and partially folded states give unique insights into their structures and dynamics and provide new understanding of protein folding and function.
Inelastic neutron scattering was used to investigate liquidlike motions and the nature of a dynamic transition in myoglobin, a small globular protein. The signature of the transition is a strong enhancement of low-frequency density fluctuations and a corresponding decrease in elastic scattering above 180 K. It is shown that the line shape of the inelastic-scattering function approximates the scaling behavior predicted for a simple liquid by mode-coupling theories in the vicinity of the liquid-glass transition.
Bone sialoprotein (BSP) and osteopontin (OPN) are two members of the SIBLING (Small Integrin-Binding LIgand, N-linked Glycoprotein) family of genetically related proteins that are clustered on human chromosome 4. We present evidence that this entire family is the result of duplication and subsequent divergent evolution of a single ancient gene. The solution structures of these two post-translationally modified recombinant proteins were solved by one dimensional proton NMR and transverse relaxation times. The polypeptide backbones of both free BSP and OPN rapidly sample an ensemble of conformations consistent with them both being completely unstructured in solution. This flexibility appears to enable these relatively small glycoproteins to rapidly associate with a number of different binding partners including other proteins as well as the mineral phase of bones and teeth. These proteins often function by bridging two proteins of fixed structures into a biologically active complex.
Recent developments in solution NMR methods have allowed for an unprecedented view of protein dynamics. Current insights into the nature of protein dynamics and their potential influence on protein structure, stability and function are reviewed. Particular emphasis is placed on the potential of fast side chain motion to report on the residual conformational entropy of proteins and how this entropy can enter into both the thermodynamic and kinetic aspects of protein function.
We present a comparative proteome analysis of the five complete eukaryotic genomes (human, Drosophila melanogaster, Caenorhabditis elegans, Saccharomyces cerevisiae, Arabidopsis thaliana), focusing on individual and multiple amino acid runs, charge and hydrophobic runs. We found that human proteins with multiple long runs are often associated with diseases; these include long glutamine runs that induce neurological disorders, various cancers, categories of leukemias (mostly involving chromosomal translocations), and an abundance of Ca(2 +) and K(+) channel proteins. Many human proteins with multiple runs function in development and/or transcription regulation and are Drosophila homeotic homologs. A large number of these proteins are expressed in the nervous system. More than 80% of Drosophila proteins with multiple runs seem to function in transcription regulation. The most frequent amino acid runs in Drosophila sequences occur for glutamine, alanine, and serine, whereas human sequences highlight glutamate, proline, and leucine. The most frequent runs in yeast are of serine, glutamine, and acidic residues. Compared with the other eukaryotic proteomes, amino acid runs are significantly more abundant in the fly. This finding might be interpreted in terms of innate differences in DNA-replication processes, repair mechanisms, DNA-modification systems, and mutational biases. There are striking differences in amino acid runs for glutamine, asparagine, and leucine among the five proteomes.
Complementary neutron- and light-scattering results on nine proteins and amino acids reveal the role of rigidity and secondary structure in determining the time- and lengthscales of low-frequency collective vibrational dynamics in proteins. These dynamics manifest in a spectral feature, known as the boson peak (BP), which is common to all disordered materials. We demonstrate that BP position scales systematically with structural motifs, reflecting local rigidity: disordered proteins appear softer than α-helical proteins; which are softer than β-sheet proteins. Our analysis also reveals a universal spectral shape of the BP in proteins and amino acid mixtures; superimposable on the shape observed in typical glasses. Uniformity in the underlying physical mechanism, independent of the specific chemical composition, connects the BP vibrations to nanometer-scale heterogeneities, providing an experimental benchmark for coarse-grained simulations, structure/rigidity relationships, and engineering of proteins for novel applications.
Collective dynamics are considered to be one of the major properties of soft materials, including biological macromolecules. We present coherent neutron scattering studies of the low-frequency vibrations, the so-called boson peak, in fully deuterated green fluorescent protein (GFP). Our analysis revealed unexpectedly low coherence of the atomic motions in GFP. This result implies a low amount of in-phase collective motion of the secondary structural units contributing to the boson peak vibrations and fast conformational fluctuations on the picosecond timescale. These observations are in contrast to earlier studies of polymers and glass-forming systems, and suggest that random or out-of-phase motions of the β-strands contribute greater than two-thirds of the intensity to the low-frequency vibrational spectra of GFP.
We present analysis of nanosecond-picosecond dynamics of Green Fluorescence Protein (GFP) using neutron scattering data obtained on three spectrometers. GFP has a β-barrel structure that differs significantly from the structure of other globular proteins and is thought to result in a more rigid local environment. Despite this difference, our analysis reveals that the dynamics of GFP are similar to dynamics of other globular proteins such as lysozyme and myoglobin. We suggest that the same general concept of protein dynamics may be applicable to all these proteins. The dynamics of dry protein are dominated by methyl group rotations, while hydration facilitates localized diffusion-like motions in the protein. The latter has an extremely broad relaxation spectrum. The nanosecond-picosecond dynamics of both dry and hydrated GFP are localized to distances of ~ 1-3.5 Å, in contrast to the longer range diffusion of hydration water.
Infrared and Raman spectra of polyethylene glycol (PEG) complexed with calcium and magnesium chlorides have been measured in aqueous solutions and in isolated complexes. Composition of the calcium complex was obtained as 4.4±0.1 (CH2CH2O unit):1 (CaCl2):4.5±0.5 (H2O) by elementary analysis. From these results, the conformation of PEG in the calcium complex has been proposed to be an alternate conbination of two conformational sequences, (-TGT-TG′T-GTT-TTG-) and (-TGT-TG′T-TGT-GTT-TTG-). For the magnesium complex, the conformation of PEG is supposed to be similar to that of crystalline PEG, with repetition of TGT for the -CH2CH2O units. The difference in the structure between the calcium and magnesium complexes was explained by the difference in the cation's size.
We report a MD simulation study of the behavior of the boson peak of a globular protein in realistic powder environments corresponding to conditions of neutron scattering studies (hydrated at 150 K, dry at 150 K, and dry at 300 K). The temperature and hydration dependence of the boson peak, an excess of inelastic scattering intensity over the harmonic background at low frequency, are in excellent agreement with neutron scattering data on powder samples of several proteins. To gain further insight into the nature of boson peak, and its relation to hydration water, we have decomposed the inelastic spectrum into contributions from the protein backbone, nonpolar side chains in the interior of the protein, and polar side chains exposed to the solvent. We find that the boson peak arises from motions distributed throughout the protein, regardless of the conditions of temperature and hydration. Furthermore, the relative contribution from each part of the protein considered shows a similar temperature and hydration dependence. This demonstrates that the damping of the boson peak upon hydration is not solely due to the damping of the water-coupled motion of exposed polar side chains, but rather propagates through the whole protein.
The conformation of β-casein A in the monomeric and thermally aggregated states has been investigated by a range of techniques. β-Casein exists as a monomer in solution at 4°C and at concentrations up to at least 3 g/dl. The molecule is flexible and exhibits a lot of segmental motion, but its secondary structure is not wholly random coil; about one-third of the polypeptide chain is ordered and the likely locations of these regions are discussed. The radius of gyration, representing the time-average distribution of the flexible chain, is 46 Å. Increasing temperature leads to aggregation of the β-casein molecules. The degree of association is very sensitive to experimental conditions, and under our conditions a 14-mer exists at 20°C. The aggregate is spherical with a radius of about 100 Å. The interior of the aggregate is relatively disordered, and the β-casein molecules remain in a largely flexible, hydrated conformation. The volume restriction of the protein molecules which occurs on association leads to some immobilization of the hydrophobic C-terminal region, which is packed toward the center of the aggregate.
We present a detailed analysis of the picosecond-to-nanosecond motions of green fluorescent protein (GFP) and its hydration water using neutron scattering spectroscopy and hydrogen/deuterium contrast. The analysis reveals that hydration water suppresses protein motions at lower temperatures (<∼200 K), and facilitates protein dynamics at high temperatures. Experimental data demonstrate that the hydration water is harmonic at temperatures <∼180-190 K and is not affected by the proteins' methyl group rotations. The dynamics of the hydration water exhibits changes at ∼180-190 K that we ascribe to the glass transition in the hydrated protein. Our results confirm significant differences in the dynamics of protein and its hydration water at high temperatures: on the picosecond-to-nanosecond timescale, the hydration water exhibits diffusive dynamics, while the protein motions are localized to <∼3 Å. The diffusion of the GFP hydration water is similar to the behavior of hydration water previously observed for other proteins. Comparison with other globular proteins (e.g., lysozyme) reveals that on the timescale of 1 ns and at equivalent hydration level, GFP dynamics (mean-square displacements and quasielastic intensity) are of much smaller amplitude. Moreover, the suppression of the protein dynamics by the hydration water at low temperatures appears to be stronger in GFP than in other globular proteins. We ascribe this observation to the barrellike structure of GFP.
Hydration water is vital for various macromolecular biological activities, such as specific ligand recognition, enzyme activity, response to receptor binding, and energy transduction. Without hydration water, proteins would not fold correctly and would lack the conformational flexibility that animates their three-dimensional structures. Motions in globular, soluble proteins are thought to be governed to a certain extent by hydration-water dynamics, yet it is not known whether this relationship holds true for other protein classes in general and whether, in turn, the structural nature of a protein also influences water motions. Here, we provide insight into the coupling between hydration-water dynamics and atomic motions in intrinsically disordered proteins (IDP), a largely unexplored class of proteins that, in contrast to folded proteins, lack a well-defined three-dimensional structure. We investigated the human IDP tau, which is involved in the pathogenic processes accompanying Alzheimer disease. Combining neutron scattering and protein perdeuteration, we found similar atomic mean-square displacements over a large temperature range for the tau protein and its hydration water, indicating intimate coupling between them. This is in contrast to the behavior of folded proteins of similar molecular weight, such as the globular, soluble maltose-binding protein and the membrane protein bacteriorhodopsin, which display moderate to weak coupling, respectively. The extracted mean square displacements also reveal a greater motional flexibility of IDP compared with globular, folded proteins and more restricted water motions on the IDP surface. The results provide evidence that protein and hydration-water motions mutually affect and shape each other, and that there is a gradient of coupling across different protein classes that may play a functional role in macromolecular activity in a cellular context.
Caseins belong to a larger group of secreted calcium phosphate-binding phosphoproteins that have a natively unfolded conformation. Nearly all members of the group are involved in aspects of calcium phosphate biology and nearly all have recognition sites for phosphorylation by the Golgi protein kinase. In the caseins these are often close together in the primary structure, forming the so-called phosphate centres. Certain highly phosphorylated phosphopeptides derived from the calcium-sensitive caseins will combine with amorphous calcium phosphate to form defined chemical complexes called calcium phosphate nanoclusters. Both the substructure of casein micelles and the partition of salts in milk can be explained quantitatively by the ability of the calcium-sensitive caseins to sequester calcium phosphate and form nanocluster-like structures. A simple stability rule for milk can be derived by applying equilibrium thermodynamics to the process of calcium phosphate sequestration. In principle, the stability rule can be extended to problems of instability encountered in milk-processing operations and to the formulation of other types of high calcium foods.
It is shown that the spectrum of excess low-energy (E∼2–10 meV) density of vibrational states in glasses found by inelastic neutron scattering is described by a log-normal law with a universal standard deviation of log E. This regularity is explained within the framework of the cluster model of glass structure.
In order to examine the properties specific to the folded protein, the effect of the conformational states on protein dynamical transition was studied by incoherent elastic neutron scattering for both wild type and a deletion mutant of staphylococcal nuclease. The deletion mutant of SNase which lacks C-terminal 13 residues takes a compact denatured structure, and can be regarded as a model of intrinsic unstructured protein. Incoherent elastic neutron scattering experiments were carried out at various temperature between 10 K and 300 K on IN10 and IN13 installed at ILL. Temperature dependence of mean-square displacements was obtained by the q-dependence of elastic scattering intensity. The measurements were performed on dried and hydrated powder samples. No significant differences were observed between wild type and the mutant for the hydrated samples, while significant differences were observed for the dried samples. A dynamical transition at ∼ 140 K observed for both dried and hydrated samples. The slopes of the temperature dependence of MSD before transition and after transition are different between wild type and the mutant, indicating the folding induces hardening. The hydration water activates a further transition at ∼ 240 K. The behavior of the temperature dependence of MSD is indistinguishable for wild type and the mutant, indicating that hydration water dynamics dominate the dynamical properties.
The dynamics of RNA contributes to its biological functions such as ligand recognition and catalysis. Using quasielastic neutron scattering spectroscopy, we show that Mg(2+) greatly increases the picosecond to nanosecond dynamics of hydrated tRNA while stabilizing its folded structure. Analyses of the atomic mean-squared displacement, relaxation time, persistence length, and fraction of mobile atoms showed that unfolded tRNA is more rigid than folded tRNA. This same result was found for a sulfonated polystyrene, indicating that the increased dynamics in Mg(2+) arises from improved charge screening of the polyelectrolyte rather than specific interactions with the folded tRNA. These results are opposite to the relationship between structural compactness and internal dynamics for proteins in which the folded state is more rigid than the denatured state. We conclude that RNA dynamics are strongly influenced by the electrostatic environment, in addition to the motions of local waters.
We describe the design and current performance of the backscattering silicon spectrometer (BASIS), a time-of-flight backscattering spectrometer built at the spallation neutron source (SNS) of the Oak Ridge National Laboratory (ORNL). BASIS is the first silicon-based backscattering spectrometer installed at a spallation neutron source. In addition to high intensity, it offers a high-energy resolution of about 3.5 μeV and a large and variable energy transfer range. These ensure an excellent overlap with the dynamic ranges accessible at other inelastic spectrometers at the SNS.
The unfolding of hen egg-white lysozyme dissolved both in D(2)O and CH(3)CH(2)OD/D(2)O was studied by Fourier Transform Infrared (FTIR) absorption spectroscopy at different protein concentrations. A detailed description of the local and global rearrangement of the secondary structure upon a temperature increase, in the range 295 to 365K, was obtained through the analysis of the amide I band. Thermodynamic parameters for the melting, and the effect of the co-solvent in determining a change in thermal stability of the protein were evaluated. The protein-protein interactions were also followed as a function of temperature: a strong dependence of the cluster stability and aggregation yield on the solvent composition was observed. Finally, FTIR spectra taken at successive time steps of the aggregation enabled intermolecular contacts to be monitored as a function of time, and kinetic information to be obtained showing that both unfolded and folded states of lysozyme act as reactants for the clustering event.
Entrapment of biomolecules in silica-derived sol-gels has grown into a vibrant area of research since it was originally demonstrated. However, accessing the consequences of entrapment on biomolecules and the gel structure remains a major challenge in characterizing these biohybrid materials. We present the first demonstration that it is possible with small-angle neutron scattering (SANS) to study the conformation of dilute proteins that are entrapped in transparent and dense sol-gels. Using deuterium-labeled green fluorescent protein (GFP) and SANS with contrast variation, we demonstrate that the scattering signatures of the sol-gel and the protein can be separated. Analysis of the scattering curves of the sol-gels using a mass-fractal model shows that the size of the colloidal silica particles and the fractal dimensions of the gels were similar in the absence and presence of protein, demonstrating that GFP did not influence the reaction pathway for the formation of the gel. The major structural difference in the gels was that the pore size was increased 2-fold in the presence of the protein. At the contrast match point for silica, the scattering signal from GFP inside the gel became distinguishable over a wide q range. Simulated scattering curves representing a monomer, end-to-end dimer, and parallel dimer of the protein were calculated and compared to the experimental data. Our results show that the most likely structure of GFP is that of an end-to-end dimer. This approach can be readily applied and holds great potential for the structural characterization of complex biohybrid and other materials.
Elastic incoherent neutron scattering has been used to study the temperature dependence of the mean-square displacements of nonexchangeable hydrogen atoms in powders of a series of homomeric polypeptides (polyglycine, polyalanine, polyphenylalanine and polyisoleucine) in comparison with myoglobin at the same hydration level (h = 0.2). The aim of the work was to measure the dynamic behavior of different amino acid residues separately and assess the contribution of each type of side chain to the anharmonic dynamics of proteins. The results provide direct experimental evidence that the first anharmonic activation, at approximately 150 K, is largely due to methyl group rotations entering the time window of the spectrometer used; however, contributions on the order of 10-20% from the motions of other groups (e.g., the phenolic ring and the methylene groups) are present. Our data also indicate that the dynamical transition occurring at approximately 230 K can be attributed, at least at the hydration level investigated, mainly to motions involving backbone fluctuations.
The influence of hydration on the nanosecond timescale dynamics of tRNA is investigated using neutron scattering spectroscopy. Unlike protein dynamics, the dynamics of tRNA is not affected by methyl group rotation. This allows for a simpler analysis of the influence of hydration on the conformational motions in RNA. We find that hydration affects the dynamics of tRNA significantly more than that of lysozyme. Both the characteristic length scale and the timescale of the conformational motions in tRNA depend strongly on hydration. Even the characteristic temperature of the so-called "dynamical transition" appears to be hydration-dependent in tRNA. The amplitude of the conformational motions in fully hydrated tRNA is almost twice as large as in hydrated lysozyme. We ascribe these differences to a more open and flexible structure of hydrated RNA, and to a larger fraction and different nature of hydrophilic sites. The latter leads to a higher density of water that makes the biomolecule more flexible. All-atom molecular-dynamics simulations are used to show that the extent of hydration is greater in tRNA than in lysozyme. We propose that water acts as a "lubricant" in facilitating enhanced motion in solvated RNA molecules.
Combining dielectric spectroscopy and neutron scattering data for hydrated lysozyme powders, we were able to identify several relaxation processes and follow protein dynamics at different hydration levels over a broad frequency and temperature range. We ascribe the main dielectric process to protein's structural relaxation coupled to hydration water and the slowest dielectric process to a larger scale protein's motions. Both relaxations exhibit a smooth, slightly super-Arrhenius temperature dependence between 300 and 180 K. The temperature dependence of the slowest process follows the main dielectric relaxation, emphasizing that the same friction mechanism might control both processes. No signs of a proposed sharp fragile-to-strong crossover at T approximately 220 K are observed in temperature dependences of these processes. Both processes show strong dependence on hydration: the main dielectric process slows down by six orders with a decrease in hydration from h approximately 0.37 (grams of water per grams of protein) to h approximately 0.05. The slowest process shows even stronger dependence on hydration. The third (fastest) dielectric relaxation process has been detected only in samples with high hydration ( h approximately 0.3 and higher). We ascribe it to a secondary relaxation of hydration water. The mechanism of the protein dynamic transition and a general picture of the protein dynamics are discussed.
The structure and dynamics of the urea-denatured B1 immunoglobulin binding domain of streptococcal protein G (GB1) has been investigated by multidimensional heteronuclear NMR spectroscopy. Complete 1H, 15N, and 13C assignments are obtained by means of sequential through-bond correlations. The nuclear Overhauser enhancement, chemical shift, and 3JHN alpha coupling constant data provide no evidence for the existence of any significant population of residual native or nonnative ordered structure. 15N relaxation measurements at 500 and 600 MHz, however, provide evidence for conformationally restricted motions in three regions of the polypeptide that correspond to the second beta-hairpin, the N-terminus of the alpha-helix, and the middle of the alpha-helix in the native protein. The time scale of these motions is longer than the apparent overall correlation time (approximately 3 ns) and could range from about 6 ns in the case of one model to between 4 microseconds and 2 ms in another; it is not possible to distinguish between these two cases with certainty because the dynamics are highly complex and hence the analysis of the time scale of this slower motion is highly model dependent. It is suggested that these three regions may correspond to nucleation sites for the folding of the GB1 domain. With the exception of the N- and C-termini, where end effects predominate, the amplitude of the subnanosecond motions, on the other hand, are fairly uniform and model independent, with an overall order parameter S2 ranging from 0.4 to 0.5.
Measurements of 15N NMR relaxation parameters have been used to characterize the backbone dynamics of folded and denatured states of the N-terminal SH3 domain from the adapter protein drk, in high salt or guanidinium chloride, respectively. Values of the spectral density function evaluated at a number of frequencies are compared. The levels of backbone dynamics in the folded protein show little variation across the molecule and are of similar magnitude to those determined previously for the folded state of the protein in exchange with an unfolded state at low salt concentrations [Farrow et al. (1995) Biochemistry 34, 868-878]. The denatured state of the domain exhibits both more extensive and more heterogeneous dynamics than the folded state. In particular the profile of the spectral density function evaluated at zero-frequency for the unfolded state of the domain indicates that residues in the middle of the protein sequence are considerably less mobile than those at the termini. These data suggest that the molecule is not behaving as an extended polymer and that concerted motions of the central portions of the molecule are occurring, consistent with a reasonably compact conformation in this region. The backbone dynamics of the folded and unfolded states were studied at two temperatures. The level of high-frequency motions in the folded molecule is largely unaffected by changes in temperature, whereas an increase in temperature results in increased high-frequency motion in the unfolded state.
Conformational changes of proteins often involve the relative motion of rigid structural domains. Normal mode analysis and molecular dynamics simulations of small globular proteins predict delocalized vibrations with frequencies below 20 cm(-1), which may be overdamped in solution due to solvent friction. In search of these modes, we have studied deuterium-exchanged myoglobin and lysozyme using inelastic neutron scattering in the low-frequency range at full and low hydration to modify the degree of damping. At room temperature, the hydrated samples exhibit a more pronounced quasielastic spectrum due to diffusive motions than the dehydrated samples. The analysis of the corresponding lineshapes suggests that water modifies mainly the amplitude, but not the characteristic time of fast protein motions. At low temperatures, in contrast, the dehydrated samples exhibit larger motional amplitudes than the hydrated ones. The excess scattering, culminating at 16 cm(-1), is suggested to reflect water-coupled librations of polar side chains that are depressed in the hydrated system by strong intermolecular hydrogen bonding. Both myoglobin and lysozyme exhibit ultra-low-frequency modes below 10 cm(-1) in the dry state, possibly related to the breathing modes predicted by harmonic analysis.
In just three years, the green fluorescent protein (GFP) from the jellyfish Aequorea victoria has vaulted from obscurity to become one of the most widely studied and exploited proteins in biochemistry and cell biology. Its amazing ability to generate a highly visible, efficiently emitting internal fluorophore is both intrinsically fascinating and tremendously valuable. High-resolution crystal structures of GFP offer unprecedented opportunities to understand and manipulate the relation between protein structure and spectroscopic function. GFP has become well established as a marker of gene expression and protein targeting in intact cells and organisms. Mutagenesis and engineering of GFP into chimeric proteins are opening new vistas in physiological indicators, biosensors, and photochemical memories.
A direct measure of intramolecular chain diffusion is obtained by the determination of triplet-triplet energy-transfer rates between a donor and an acceptor chromophore attached at defined points on a polypeptide chain. Single exponential kinetics of contact formation are observed on the nanosecond time scale for polypeptides in which donor and acceptor are linked by repeating units of glycine and serine residues. The rates depend on the number of peptide bonds (N) separating donor and acceptor and show a maximum for the shortest peptides (N = 3) with a time constant (tau = 1/k) of 20 ns. This sets an upper limit for the speed of formation of the first side-chain contacts during protein folding.
A major challenge in the post-genome era will be determination of the functions of the encoded protein sequences. Since it is generally assumed that the function of a protein is closely linked to its three-dimensional structure, prediction or experimental determination of the library of protein structures is a matter of high priority. However, a large proportion of gene sequences appear to code not for folded, globular proteins, but for long stretches of amino acids that are likely to be either unfolded in solution or adopt non-globular structures of unknown conformation. Characterization of the conformational propensities and function of the non-globular protein sequences represents a major challenge. The high proportion of these sequences in the genomes of all organisms studied to date argues for important, as yet unknown functions, since there could be no other reason for their persistence throughout evolution. Clearly the assumption that a folded three-dimensional structure is necessary for function needs to be re-examined. Although the functions of many proteins are directly related to their three-dimensional structures, numerous proteins that lack intrinsic globular structure under physiological conditions have now been recognized. Such proteins are frequently involved in some of the most important regulatory functions in the cell, and the lack of intrinsic structure in many cases is relieved when the protein binds to its target molecule. The intrinsic lack of structure can confer functional advantages on a protein, including the ability to bind to several different targets. It also allows precise control over the thermodynamics of the binding process and provides a simple mechanism for inducibility by phosphorylation or through interaction with other components of the cellular machinery. Numerous examples of domains that are unstructured in solution but which become structured upon binding to the target have been noted in the areas of cell cycle control and both transcriptional and translational regulation, and unstructured domains are present in proteins that are targeted for rapid destruction. Since such proteins participate in critical cellular control mechanisms, it appears likely that their rapid turnover, aided by their unstructured nature in the unbound state, provides a level of control that allows rapid and accurate responses of the cell to changing environmental conditions.
The partly folded state of apomyoglobin at pH 4 represents an excellent model for an obligatory kinetic folding intermediate. The structure and dynamics of this intermediate state have been extensively examined using NMR spectroscopy. Secondary chemical shifts, (1)H-(1)H NOEs, and amide proton temperature coefficients have been used to probe residual structure in the intermediate state, and NMR relaxation parameters T(1) and T(2) and ¿(1)H¿-(15)N NOE have been analyzed using spectral densities to correlate motion of the polypeptide chain with these structural observations. A significant amount of helical structure remains in the pH 4 state, indicated by the secondary chemical shifts of the (13)C(alpha), (13)CO, (1)H(alpha), and (13)C(beta) nuclei, and the boundaries of this helical structure are confirmed by the locations of (1)H-(1)H NOEs. Hydrogen bonding in the structured regions is predominantly native-like according to the amide proton chemical shifts and their temperature dependence. The locations of the A, G, and H helix segments and the C-terminal part of the B helix are similar to those in native apomyoglobin, consistent with the early, complete protection of the amides of residues in these helices in quench-flow experiments. These results confirm the similarity of the equilibrium form of apoMb at pH 4 and the kinetic intermediate observed at short times in the quench-flow experiment. Flexibility in this structured core is severely curtailed compared with the remainder of the protein, as indicated by the analysis of the NMR relaxation parameters. Regions with relatively high values of J(0) and low values of J(750) correspond well with the A, B, G, and H helices, an indication that nanosecond time scale backbone fluctuations in these regions of the sequence are restricted. Other parts of the protein show much greater flexibility and much reduced secondary chemical shifts. Nevertheless, several regions show evidence of the beginnings of helical structure, including stretches encompassing the C helix-CD loop, the boundary of the D and E helices, and the C-terminal half of the E helix. These regions are clearly not well-structured in the pH 4 state, unlike the A, B, G, and H helices, which form a native-like structured core. However, the proximity of this structured core most likely influences the region between the B and F helices, inducing at least transient helical structure.
The mutant proteins that cause polyglutamine disease bind CREB-binding protein (CBP), a key transcriptional coactivator for neuronal survival factors. This results in a loss of CBP-dependent transcription and may account for the neuronal degeneration associated with these diseases.
Proteins can exist in a trinity of structures: the ordered state, the molten globule, and the random coil. The five following examples suggest that native protein structure can correspond to any of the three states (not just the ordered state) and that protein function can arise from any of the three states and their transitions. (1) In a process that likely mimics infection, fd phage converts from the ordered into the disordered molten globular state. (2) Nucleosome hyperacetylation is crucial to DNA replication and transcription; this chemical modification greatly increases the net negative charge of the nucleosome core particle. We propose that the increased charge imbalance promotes its conversion to a much less rigid form. (3) Clusterin contains an ordered domain and also a native molten globular region. The molten globular domain likely functions as a proteinaceous detergent for cell remodeling and removal of apoptotic debris. (4) In a critical signaling event, a helix in calcineurin becomes bound and surrounded by calmodulin, thereby turning on calcineurin's serine/threonine phosphatase activity. Locating the calcineurin helix within a region of disorder is essential for enabling calmodulin to surround its target upon binding. (5) Calsequestrin regulates calcium levels in the sarcoplasmic reticulum by binding approximately 50 ions/molecule. Disordered polyanion tails at the carboxy terminus bind many of these calcium ions, perhaps without adopting a unique structure. In addition to these examples, we will discuss 16 more proteins with native disorder. These disordered regions include molecular recognition domains, protein folding inhibitors, flexible linkers, entropic springs, entropic clocks, and entropic bristles. Motivated by such examples of intrinsic disorder, we are studying the relationships between amino acid sequence and order/disorder, and from this information we are predicting intrinsic order/disorder from amino acid sequence. The sequence-structure relationships indicate that disorder is an encoded property, and the predictions strongly suggest that proteins in nature are much richer in intrinsic disorder than are those in the Protein Data Bank. Recent predictions on 29 genomes indicate that proteins from eucaryotes apparently have more intrinsic disorder than those from either bacteria or archaea, with typically > 30% of eucaryotic proteins having disordered regions of length > or = 50 consecutive residues.
Natively unfolded or intrinsically unstructured proteins constitute a unique group of the protein kingdom. The evolutionary persistence of such proteins represents strong evidence in the favor of their importance and raises intriguing questions about the role of protein disorders in biological processes. Additionally, natively unfolded proteins, with their lack of ordered structure, represent attractive targets for the biophysical studies of the unfolded polypeptide chain under physiological conditions in vitro. The goal of this study was to summarize the structural information on natively unfolded proteins in order to evaluate their major conformational characteristics. It appeared that natively unfolded proteins are characterized by low overall hydrophobicity and large net charge. They possess hydrodynamic properties typical of random coils in poor solvent, or premolten globule conformation. These proteins show a low level of ordered secondary structure and no tightly packed core. They are very flexible, but may adopt relatively rigid conformations in the presence of natural ligands. Finally, in comparison with the globular proteins, natively unfolded polypeptides possess 'turn out' responses to changes in the environment, as their structural complexities increase at high temperature or at extreme pH.