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ABSTRACT: PAPf39 is a 39 residue peptide fragment from human prostatic acidic phosphatase that forms amyloid fibrils in semen. These fibrils have been implicated in facilitating HIV transmission. To enablestructural studies of PAPf39 by NMR spectroscopy, efficient methods allowing the production of milligram quantities of isotopically labeled peptide are essential. Here, we report the high-yield expression, as a fusion to ubiquitin at the N-terminus and an intein at the C-terminus, and purification of uniformly labeled (13)C- and (15)N-labeled PAPf39peptide. Thisallows the study of the PAPf39 monomer conformational ensemble by NMR spectroscopy. To this end, we performed the NMR chemical shift assignment of the PAPf39 peptide in the monomeric state at low pH.
Protein Expression and Purification 01/2013; · 1.59 Impact Factor
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ABSTRACT: Residual structure in the unfolded state of a protein may play a crucial role in folding and stability. In the present study, using an all (heavy)-atom structure based model and replica exchange molecular dynamics simulations, we explored the folding landscape of the third fibronectin type III domain from tenascin-C (TNfn3). Specifically, both the wild type (WT) and a variant with two additional amino acids Gly-Leu (GL), at the C-terminus, WT(+GL), were studied. We found that although both domains of TNfn3 are topologically frustrated, the early formation of the native contacts from the C-terminal end of (WT(+GL)) causes more "backtracking" than in the WT. As a result, the WT exhibits a two-state folding behavior with a broad transition-state ensemble, whereas the WT(+GL) folds through a metastable intermediate state. Furthermore, our study confirmed that the core of both proteins is conformationally heterogeneous and non-compact, and folds late mainly due to backtracking of the part of the core. Finally, in agreement with the previous experimental studies our results clearly demonstrated distinct thermodynamic behavior of the two proteins with WT(+GL) being more stable.
The Journal of Physical Chemistry B 12/2012; · 3.70 Impact Factor
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ABSTRACT: PAPf39, a 39 residue peptide fragment from human prostatic acidic phosphatase, has been shown to form amyloid fibrils in semen (SEVI), which increase HIV infectivity by up to five orders of magnitude. The sequence of the PAPf39 fibrillar core was identified using HDXMS and protease protection assays. The central and C-terminal regions are highly protected from HDX and proteolytic cleavage and, thus, are part of the fibrillar core. Conversely, the N-terminal region is unprotected from HDX and proteolytic cleavage, suggesting that it is exposed and not part of the fibrillar core. This finding was tested using two N-terminal truncated variants, PAPf39Δ1-8 and PAPf39Δ1-13. Both variants formed amyloid fibrils at neutral pH. However, these variants showed a markedly different pH dependence of fibril formation than PAPf39. PAPf39 fibrils can form at pH 7.7, but not at pH 5.5 and pH 2.5, while both N-terminal truncated variants can form fibrils at these pH values. This suggests that the N-terminal region is not necessary for fibril formation but modulates the pH dependence of PAPf39 fibril formation. PAPf39Δ1-8 and PAPf39Δ1-13 are capable of seeding PAPf39 fibril formation at neutral pH, suggesting that these variants are structurally compatible with PAPf39. Yet, no mixed fibril formation occurs between truncated variants and PAPf39 at low pH. This suggests that pH affects the PAPf39 monomer conformational ensemble, which is supported by far-UV CD spectroscopy. A conceptual model describing the pH dependence of PAPf39 aggregation is proposed and provides potential biological implications.
Biochemistry 12/2012; · 3.42 Impact Factor
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ABSTRACT: Trimethylamine N-oxide (TMAO) is a naturally occurring protecting osmolyte that stabilizes the folded state of proteins and also counteracts the destabilizing effect of urea on protein stability. Experimentally, it has been inferred that TMAO is preferentially excluded from the vicinity of protein surfaces. Here, we combine computer modeling and experimental measurements to gain an understanding of the mechanism of the protecting effect of TMAO on proteins. We have developed an all-atom molecular model for TMAO that captures the exclusion of TMAO from model compounds and protein surfaces, as a consequence of incorporating realistic TMAO-water interactions through osmotic pressure measurements. Osmotic pressure measurements also suggest no significant attraction between urea and TMAO molecules in solution. To obtain an accurate potential for molecular simulations of protein stability in TMAO solutions, we have explored different ways of parametrizing the protein/osmolyte and osmolyte/osmolyte interactions by scaling charges and the strength of Lennard-Jones interactions and carried out equilibrium folding experiments of Trp-cage miniprotein in the presence of TMAO to guide the parametrization. Our calculations suggest a general principle for preferential interaction behavior of cosolvents with protein surfaces-preferentially excluded osmolytes have repulsive self-interaction given by osmotic coefficient ϕ > 1, while denaturants, in addition to having attractive interactions with the proteins, have favorable self-interaction given by osmotic coefficient ϕ < 1, to enable preferential accumulation in the vicinity of proteins.
The Journal of Physical Chemistry B 09/2012; 116(40):12095-104. · 3.70 Impact Factor
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ABSTRACT: S100B is a member of the S100 subfamily of EF-hand proteins that has been implicated in malignant melanoma and neurodegenerative conditions such as Alzheimer's disease and Parkinson's disease. Calcium-induced conformational changes expose a hydrophobic binding cleft, facilitating interactions with a wide variety of nuclear, cytoplasmic, and extracellular target proteins. Previously, peptides derived from CapZ, p53, NDR, HDM2, and HDM4 have been shown to interact with S100B in a calcium-dependent manner. However, the thermodynamic and kinetic basis of these interactions remains largely unknown. To gain further insight, we screened these peptides against the S100B protein using isothermal titration calorimetry and nuclear magnetic resonance. All peptides were found to have binding affinities in the low micromolar to nanomolar range. Binding-induced changes in the line shapes of S100B backbone (1)H and (15)N resonances were monitored to obtain the dissociation constants and the kinetic binding parameters. The large microscopic K(on) rate constants observed in this study (≥1 × 10(7) M(-1) s(-1)) suggest that S100B utilizes a "fly casting mechanism" in the recognition of these peptide targets.
Biochemistry 08/2012; 51(36):7189-201. · 3.42 Impact Factor
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ABSTRACT: Statistical analysis of the residue separation between a pair of ionizable side chains within 4 Å of each other was performed on a set of 1560 non-homologous PDB structures. We found that the frequency of pairs of like charges (i.e., pairs consisting of acidic residues Asp and Glu or pairs consisting of basic residues Arg and Lys) is two orders of magnitude lower than the pairs of oppositely charged residues (salt-bridges). We also found that for pairs of like charges the distribution is skewed dramatically towards short residue separation (<3). On the basis of these observations, we hypothesize that at short residue separation the repulsion between charges does not contribute much to the protein stability and the effects are largely dominated by the long range charge-charge interactions with other ionizable groups in the protein molecule. To test this hypothesis, we incorporated various pairs of charged residues at position 63 and 64 of ubiquitin and compared the stabilities of these variants. We also performed calculations of the expected changes in the charge-charge interactions. A very good correlation between experimental changes in the stability of ubiquitin variants, and changes in the energy of charge-charge interactions provides support for the hypothesis that a pair of ionizable residues next to each other in sequence modulates protein stability via long range charge-charge interactions with the rest of the protein.
Proteins Structure Function and Bioinformatics 12/2011; 79(12):3494-9. · 3.39 Impact Factor
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ABSTRACT: Optimization of surface exposed charge-charge interactions in the native state has emerged as an effective means to enhance protein stability; but the effect of electrostatic interactions on the kinetics of protein folding is not well understood. To investigate the kinetic consequences of surface charge optimization, we characterized the folding kinetics of a Fyn SH3 domain variant containing five amino acid substitutions that was computationally designed to optimize surface charge-charge interactions. Our results demonstrate that this optimized Fyn SH3 domain is stabilized primarily through an eight-fold acceleration in the folding rate. Analyses of the constituent single amino acid substitutions indicate that the effects of optimization of charge-charge interactions on folding rate are additive. This is in contrast to the trend seen in folded state stability, and suggests that electrostatic interactions are less specific in the transition state compared to the folded state. Simulations of the transition state using a coarse-grained chain model show that native electrostatic contacts are weakly formed, thereby making the transition state conducive to nonspecific, or even nonnative, electrostatic interactions. Because folding from the unfolded state to the folding transition state for small proteins is accompanied by an increase in charge density, nonspecific electrostatic interactions, that is, generic charge density effects can have a significant contribution to the kinetics of protein folding. Thus, the interpretation of the effects of amino acid substitutions at surface charged positions may be complicated and consideration of only native-state interactions may fail to provide an adequate picture.
Proteins Structure Function and Bioinformatics 11/2011; 80(3):858-70. · 3.39 Impact Factor
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ABSTRACT: The Trp-cage miniprotein is a 20 amino acid peptide that exhibits many of the properties of globular proteins. In this protein, the hydrophobic core is formed by a buried Trp side chain. The folded state is stabilized by an ion pair between aspartic acid and an arginine side chain. The effect of protonating the aspartic acid on the Trp-cage miniprotein folding/unfolding equilibrium is studied by explicit solvent molecular dynamics simulations of the protein in the charged and protonated Asp9 states. Unbiased Replica Exchange Molecular Dynamics (REMD) simulations, spanning a wide temperature range, are carried out to the microsecond time scale, using the AMBER99SB forcefield in explicit TIP3P water. The protein structural ensembles are studied in terms of various order parameters that differentiate the folded and unfolded states. We observe that in the folded state the root mean square distance (rmsd) from the backbone of the NMR structure shows two highly populated basins close to the native state with peaks at 0.06 nm and 0.16 nm, which are consistent with previous simulations using the same forcefield. The fraction of folded replicas shows a drastic decrease because of the absence of the salt bridge. However, significant populations of conformations with the arginine side chain exposed to the solvent, but within the folded basin, are found. This shows the possibility to reach the folded state without formation of the ion pair. We also characterize changes in the unfolded state. The equilibrium populations of the folded and unfolded states are used to characterize the thermodynamics of the system. We find that the change in free energy difference due to the protonation of the Asp amino acid is 3 kJ mol(-1) at 297 K, favoring the charged state, and resulting in ΔpK(1) = 0.5 units for Asp9. We also study the differences in the unfolded state ensembles for the two charge states and find significant changes at low temperature, where the protonated Asp side chain makes multiple hydrogen bonds to the protein backbone.
Physical Chemistry Chemical Physics 07/2011; 13(38):17056-63. · 3.57 Impact Factor
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ABSTRACT: It is well known that nonpolar residues are largely buried in the interior of proteins, whereas polar and ionizable residues tend to be more localized on the protein surface where they are solvent exposed. Such a distribution of residues between surface and interior is well understood from a thermodynamic point: nonpolar side chains are excluded from the contact with the solvent water, whereas polar and ionizable groups have favorable interactions with the water and thus are preferred at the protein surface. However, there is an increasing amount of information suggesting that polar and ionizable residues do occur in the protein core, including at positions that have no known functional importance. This is inconsistent with the observations that dehydration of polar and in particular ionizable groups is very energetically unfavorable. To resolve this, we performed a detailed analysis of the distribution of fractional burial of polar and ionizable residues using a large set of ˜2600 nonhomologous protein structures. We show that when ionizable residues are fully buried, the vast majority of them form hydrogen bonds and/or salt bridges with other polar/ionizable groups. This observation resolves an apparent contradiction: the energetic penalty of dehydration of polar/ionizable groups is paid off by favorable energy of hydrogen bonding and/or salt bridge formation in the protein interior. Our conclusion agrees well with the previous findings based on the continuum models for electrostatic interactions in proteins.
Proteins Structure Function and Bioinformatics 07/2011; 79(7):2027-32. · 3.39 Impact Factor
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ABSTRACT: Understanding the origins of cooperativity in proteins remains an important topic in protein folding. This study describes experimental folding/unfolding equilibrium and kinetic studies of the engineered protein Ubq-UIM, consisting of ubiquitin (Ubq) fused to the sequence of the ubiquitin interacting motif (UIM) via a short linker. Urea-induced folding/unfolding profiles of Ubq-UIM were monitored by far-UV circular dichroism and fluorescence spectroscopies and compared to those of the isolated Ubq domain. It was found that the equilibrium data for Ubq-UIM is inconsistent with a two-state model. Analysis of the kinetics of folding shows similarity in the folding transition state ensemble between Ubq and Ubq-UIM, suggesting that formation of Ubq domain is independent of UIM. The major contribution to the stabilization of Ubq-UIM, relative to Ubq, was found to be in the rates of unfolding. Moreover, it was found that the kinetic m-values for Ubq-UIM unfolding, monitored by different probes (far-UV circular dichroism and fluorescence spectroscopies), are different; thereby, further supporting deviations from a two-state behavior. A thermodynamic linkage model that involves four states was found to be applicable to the urea-induced unfolding of Ubq-UIM, which is in agreement with the previous temperature-induced unfolding study. The applicability of the model was further supported by site-directed variants of Ubq-UIM that have altered stabilities of Ubq/UIM interface and/or stabilities of individual Ubq- and UIM-domains. All variants show increased cooperativity and one variant, E43N_Ubq-UIM, appears to behave very close to an equilibrium two-state.
Biophysical chemistry 05/2011; 159(1):58-65. · 2.28 Impact Factor
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ABSTRACT: We report the application of pressure perturbation calorimetry (PPC) to study unfolded proteins. Using PPC we have measured the temperature dependence of the thermal expansion coefficient, α(T), in the unfolded state of apocytochrome C and reduced BPTI. We have shown that α(T) is a nonlinear function and decreases with increasing temperature. The decrease is most significant in the low (2-55 °C) temperature range. We have also tested an empirical additivity approach to predict α(T) of unfolded state from the amino acid sequence using α(T) values for individual amino acids. A comparison of the experimental and calculated functions shows a very good agreement, both in absolute values of α(T) and in its temperature dependence. Such an agreement suggests the applicability of using empirical calculations to predict α(T) of any unfolded protein.
The Journal of Physical Chemistry B 12/2010; 114(49):16166-70. · 3.70 Impact Factor
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ABSTRACT: This study describes the thermodynamic characterization of a Ubq-UIM fusion construct (Ubq-UIM), designed from the ubiquitin-UIM interaction system, to determine whether it exhibits cooperativity of folding. The Ubq-UIM fusion constructs exhibit higher stability than the core Ubq molecule, consistent with the finding that the UIM helix is docked to Ubq. Temperature-induced unfolding profiles of Ubq-UIM were monitored by DSC and far-UV and near-UV CD spectroscopies. Ubq-UIM appears to exhibit cooperative unfolding as indicated by results of global fits of a two-state model to far- and near-UV CD and DSC thermal unfolding data. The cooperativity of Ubq-UIM unfolding was further tested by the amino acid substitutions that selectively stabilize or destabilize Ubq, UIM, and/or the interface. The effects of these substitutions on the thermodynamic properties of Ubq-UIM are described well by a thermodynamic model for cooperativity in proteins. In particular, a substitution that lowered the stability of the Ubq-UIM interface indeed led to a decrease in cooperativity.
Biochemistry 10/2010; 49(39):8455-67. · 3.42 Impact Factor
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ABSTRACT: It is well established that calcium binding leads to conformational changes in S100 proteins. These conformational changes are thought to activate the protein and render a protein conformation that is capable of binding other proteins. The basic quaternary structural motif of S100 proteins is a homodimer, however there is little information if higher order non-covalent oligomers are also formed and whether these oligomers are of functional relevance. To this end we performed equilibrium analytical ultracentrifugation experiments for 16 S100 proteins (S100A1, S100A2, S100A3, S100A4, S100A5, S100A6, S100A7, S100A8, S100A9, S100A10, S100A11, S100A12, S100A13, S100B, S100P, and S100Z) under reducing conditions in the absence and presence of calcium ions. We show that the addition of calcium promotes the formation of tetrameric structures which could be further enhanced under in vivo conditions where there is an additional effect of molecular crowding.
Biophysical chemistry 10/2010; 151(3):181-6. · 2.28 Impact Factor
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ABSTRACT: The thermodynamic properties of unfolding of the Trp-cage mini protein in the presence of various concentrations of urea have been characterized using temperature-induced unfolding monitored by far-UV circular dichroism spectroscopy. Analysis of the data using a two-state model allowed the calculation of the Gibbs energy of unfolding at 25 degrees C as a function of urea concentration. This in turn was analyzed by the linear extrapolation model that yielded the dependence of Gibbs energy on urea concentration, i.e. the m-value for Trp-cage unfolding. The m-value obtained from the experimental data, as well as the experimental heat capacity change upon unfolding, were correlated with the structural parameters derived from the three dimensional structure of Trp-cage. It is shown that the m-value can be predicted well using a transfer model, while the heat capacity changes are in very good agreement with the empirical models based on model compounds studies. These results provide direct evidence that Trp-cage, despite its small size, is an excellent model for studies of protein unfolding and provide thermodynamic data that can be used to compare with atomistic computer simulations.
Proteins Structure Function and Bioinformatics 05/2010; 78(6):1376-81. · 3.39 Impact Factor
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ABSTRACT: Ubiquitin-interacting motifs (UIMs) are an important class of protein domains that interact with ubiquitin or ubiquitin-like proteins. These approximately 20-residue-long domains are found in a variety of ubiquitin receptor proteins and serve as recognition modules towards intracellular targets, which may be individual ubiquitin subunits or polyubiquitin chains attached to a variety of proteins. Previous structural studies of interactions between UIMs and ubiquitin have shown that UIMs adopt an extended structure of a single alpha-helix, containing a hydrophobic surface with a conserved sequence pattern that interacts with key hydrophobic residues on ubiquitin. In light of this large body of structural studies, details regarding the presence and the roles of structural dynamics and plasticity are surprisingly lacking. In order to better understand the structural basis of ubiquitin-UIM recognition, we have characterized changes in the structure and dynamics of ubiquitin upon binding of a UIM domain from the yeast Vps27 protein. The solution structure of a ubiquitin-UIM fusion protein designed to study these interactions is reported here and found to consist of a well-defined ubiquitin core and a bipartite UIM helix. Moreover, we have studied the plasticity of the docking interface, as well as global changes in ubiquitin due to UIM binding at the picoseconds-to-nanoseconds and microseconds-to-milliseconds protein motions by nuclear magnetic resonance relaxation. Changes in generalized-order parameters of amide groups show a distinct trend towards increased structural rigidity at the UIM-ubiquitin interface relative to values determined in unbound ubiquitin. Analysis of (15)N Carr-Purcell-Meiboom-Gill relaxation dispersion measurements suggests the presence of two types of motions: one directly related to the UIM-binding interface and the other induced to distal parts of the protein. This study demonstrates a case where localized interactions among protein domains have global effects on protein motions at timescales ranging from picoseconds to milliseconds.
Journal of Molecular Biology 03/2010; 396(4):1128-44. · 4.00 Impact Factor
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ABSTRACT: PAPf39 is a 39-residue peptide fragment from the sequence of human prostatic acidic phosphatase. This peptide was shown to form amyloid-like fibrils, which have been implicated in facilitating semen-mediated HIV transmission. Thus understanding molecular details of PAPf39 peptide fibril formation may aid in elucidating the mechanism of how PAPf39 fibrils are involved in HIV etiology. To this end, the kinetics of PAPf39 peptide fibrillization was studied using a battery of biophysical methods (atomic force microscopy, ThT fluorescence assays, far-UV circular dichroism spectroscopy, deep-UV resonance Raman spectroscopy, size exclusion chromatography, analytical ultracentrifugation, and small-angle X-ray scattering). It has been shown that fibril formation follows a nucleation-dependent elongation mechanism. Several critical factors for fibrillization have been identified. It was shown that agitation and/or seeding is required for fibril formation at 37 degrees C and neutral pH, with an additional requirement of a salt concentration above approximately 100 mM. Fibril formation by the PAPf39 peptide is inhibited by low pH or by low salt concentration at neutral pH. These observations suggest that the nucleation and fibrillization of the PAPf39 peptide are a tug-of-war between the interactions formed upon agitation and the electrostatic interactions, modulated by pH and salt concentration.
Biochemistry 11/2009; 48(48):11582-91. · 3.42 Impact Factor
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ABSTRACT: Both pressure and temperature are important environmental variables, and to obtain a complete understanding of the mechanisms of protein folding, it is necessary to determine how protein stability is dependent on these fundamental thermodynamic parameters. Although the temperature dependence of protein stability has been widely explored, the dependence of protein stability on pressure is not as well studied. In this paper, we report the results of the direct thermodynamic determination of the change in specific volume (DeltaV/V) upon protein unfolding, which defines the pressure dependence of protein stability, for five model proteins (ubiquitin, eglin c, ribonuclease A, lysozyme, and cytochrome c). We have shown that the specific volumetric changes upon unfolding for four of the proteins (ubiquitin, eglin c, ribonuclease A, and lysozyme) appear to converge to a common value at high temperatures. Analysis of various contributions to the change in volume upon protein unfolding allowed us to put forth the hypothesis that the change in volume due to hydration is very close to zero at this temperature, such that DeltaV/V is defined largely by the total volume of cavities and voids within a protein, and that this is a universal property of all small globular proteins without prosthetic groups. To test this hypothesis, additional experiments were performed with variants of eglin c that had site-directed substitutions at two buried positions, to create an additional cavity in the protein core. The results of these experiments, coupled with the structural analysis of cytochrome c showing a lower packing density compared to those of the other four proteins, provided further support for the hypothesis. Finally, we have shown that the deviation of the high-temperature DeltaV value of a given protein from the convergence value can be used to determine the size of the excess cavities in globular proteins.
Biochemistry 10/2009; 48(46):10846-51. · 3.42 Impact Factor
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ABSTRACT: A library of cold shock protein B mutant variants was employed to examine differences in protein binding behavior in ion exchange and multimodal chromatography. Single site mutations introduced at charged amino acids on the protein surface resulted in a homologous protein set with varying charge density and distribution. The retention times of the mutants varied significantly during linear gradient chromatography in both systems. The majority of the proteins were more strongly retained on the multimodal cation exchange resin as compared to the traditional cation exchanger. Further, the elution order of the mutants on the multimodal resin was different from that obtained with the ion exchanger. Quantitative structure-property relationship models generated using a support vector regression technique were shown to provide good predictions for the retention times of protein mutants on the multimodal resin. A coarse-grained ligand docking package was employed to examine the various interactions between the proteins and ligands in free solution. The multimodal ligand was shown to utilize multiple interaction types to achieve stronger retention on the protein surface. The use of this protein library in concert with the qualitative and quantitative analyses presented in this paper provides an improved understanding of protein behavior in multimodal chromatographic systems.
Journal of chromatography. A 09/2009; 1217(2):191-8. · 4.19 Impact Factor
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ABSTRACT: The S100 proteins make up a family of dimeric calcium binding proteins that function in response to changing calcium levels. Several S100 binding proteins have been identified; however, the exact biological functions of the S100 proteins are largely unknown as there are several factors which modulate their functions. To address these issues, the specificity of binding of representative members of the human S100 proteins to short N-terminal peptides of annexin I (AnI) and annexin II (AnII) was investigated under controlled experimental conditions. AnI and AnII have been shown previously to interact with S100A11 and S100A10, respectively. This provided a unique opportunity to determine their binding specificity with the other members of the human S100 protein family. It was found that AnI binds S100A6 or S100A11 while AnII binds S100A10 or S100A11. This is the first report of the interaction between S100A6 and AnI. The fact that AnI and AnII bind to selected members of the S100 protein family shows that these interactions are specific and that the mode of binding is different from that of calmodulin, as it was found not to bind AnI or AnII. From the analysis of the thermodynamics of interactions, the binding seems to be entropically driven. It was found that both AnI and AnII undergo a coil-to-helix transition upon binding to their respective binding partners. The observation that there is an overlap in functionality is not surprising due to considerable sequence homology between S100 protein family members. In fact, the functional overlap can explain previous failures of S100 knockout constructs to show any detectable changes in phenotype despite numerous implications of these proteins in important cellular processes.
Biochemistry 04/2009; 48(12):2788-98. · 3.42 Impact Factor
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ABSTRACT: Here, we report the application of a computational approach that allows the rational design of enzymes with enhanced thermostability while retaining full enzymatic activity. The approach is based on the optimization of the energy of charge-charge interactions on the protein surface. We experimentally tested the validity of the approach on 2 human enzymes, acylphosphatase (AcPh) and Cdc42 GTPase, that differ in size (98 vs. 198-aa residues, respectively) and tertiary structure. We show that the designed proteins are significantly more stable than the corresponding WT proteins. The increase in stability is not accompanied by significant changes in structure, oligomerization state, or, most importantly, activity of the designed AcPh or Cdc42. This success of the design methodology suggests that it can be universally applied to other enzymes, on its own or in combination with the other strategies based on redesign of the interactions in the protein core.
Proceedings of the National Academy of Sciences 03/2009; 106(8):2601-6. · 9.68 Impact Factor
