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Thioflavin T as a Molecular Rotor: Fluorescent Properties of Thioflavin T in Solvents with Different Viscosity
Abstract
The effect of solvent viscosity on thioflavin T (ThT) fluorescent properties is analyzed to understand the molecular mechanisms of the characteristic increase in ThT fluorescence intensity accompanying its incorporation into the amyloid-like fibrils. To this end, the dependencies of the ThT quantum yield and fluorescence lifetime on temperature and glycerol content in the water-glycerol mixtures are studied. It has been found that fluorescent properties of ThT are typical for the specific class of fluorophores known as molecular rotors. It has been established that the low ThT fluorescence intensity in the solvents with low viscosity is caused by the nonradiative deactivation of the excited state associated with the torsional motion of the ThT benzthiazole and aminobenzene rings relative to each other, which results in the transition of ThT molecule to nonfluorescent twisted internal charge transfer (TICT) state. The rate of this process is determined by the solvent viscosity, whereas the emission does occur from the nonequilibrium locally excited (LE) state. High polarization degree of the ThT fluorescence (P = 0.45) observed for glycerol solutions of different viscosity confirms the nonequilibrium character of the emission from the LE state and testifies that rotational correlation time of the whole molecule is considerably greater than the time required to accomplish transition to the nonfluorescent TICT state. Torsional movements of the ThT fragments take place in the same temporal interval as solvent relaxation, which leads to nonexponential fluorescence decay of the dye in viscous solvents. This photophysical model successfully explains the fluorescent properties of ThT in solvents with different viscosities. The model is confirmed by the results of the quantum-chemical calculations, which showed that energy minimum for the ground state of ThT corresponds to conformation with torsional angle phi = 37 degrees between the benzthiazole and aminobenzene rings and in the excited-state twisted conformation of ThT with phi = 90 degrees has minimal energy. These data support the idea that the reason for the characteristic increase in the ThT fluorescence intensity accompanying its incorporation into the amyloid fibrils is determined by the rigidity of the dye environment, which prevents the rotation of the benzthiazole ring relative to the aminobenzene ring in the excited state.
... The steady-state absorption and emission spectra recorded in pure solvents was found to match with previous reports. 15,40,43 The emission spectra shown in Figure 1 are not corrected for their absorbance at excitation wavelength, and therefore, their intensity should not be directly compared. Moreover, we are reiterating that the tert-butanol−glycerol mixture was studied at 30°C instead of 25°C unlike the other solvent mixtures. ...
... Before further exploration, it is necessary to discuss the shift in the spectra. The excited-state deactivation process of ThT involves the formation of the TICT state from the LE state, which is viscosity-controlled. 1,14,15,18,40 However, the excitation from the ground to the excited state and the subsequent deactivation process of the molecules are also influenced by the polarity of the medium. 40−42 The dielectric constant of the solvents studied ranged from 10.9 (tert-butanol) to 46.5 (glycerol). ...
... To validate our measurements, we compared the values in pure solvents with the reported values and found them to be comparable. 1,15,40,41 φ ThT gradually increased with increasing concentration of glycerol in each mixture, which correlates with the increase in fluorescence emission intensity shown in Figure 1. However, the specific variation pattern differs for each case, and φ ThT exhibits a nonlinear relationship with the glycerol concentration across all systems. ...
Molecular rotor dye thioflavin T (ThT) is almost nonfluorescent in low-viscosity solvents but highly fluorescent when bound to amyloid fibrils. This unique property arises from the rotation of the dimethylaniline moiety relative to the benzothiazole moiety in the excited state, which drives the dye from an emissive locally excited state to a twisted intramolecular charge-transfer state. This process is viscosity-controlled, and therefore, we can use the quantum yield of ThT to assess the viscosity of the environment. In this study, we have investigated the quantum yield of ThT (φThT) in various compositions of six alcoholic solvent mixtures of glycerol with methanol, ethanol, n-propanol, iso-propanol, n-butanol, and tert-butanol. We have proposed an empirical model using φThT as a function of the mole fraction of glycerol to estimate the interaction parameters between the components of the solvent mixtures. This analysis allowed us to predict the extent of nonideality of the solvent mixtures. The Förster–Hoffmann- and Loutfy–Arnold-type power law relationship was established between the quantum yield of ThT and bulk viscosity for solvent mixtures of methanol, ethanol, n-butanol, and tert-butanol with glycerol, and it was found to be similar in nature in all the four mixtures. Applying this knowledge, we proposed a methodology to quantify and predict the bulk viscosity coefficient values of several compositions of n-propanol–glycerol and iso-propanol–glycerol mixtures which have not been previously documented.
... Jyotirmayee et al. achieved a nearly 2100fold enhancement of ThT fluorescence when binding it to human telomeric G4 in the presence of K + (13). Furthermore, it was proposed that the reason for ThT fluorescence excitation was that G4 provided ThT with suitable insertion room via G-quartet formation, allowing its benzothiazole (BZT) and dimethylbenzyl (DMAB) rings to be on the same plane, restricting the free rotation of C-C (14) to limit the transformation ThT state from locally excited to twisted intramolecular charge transfer (15). Although G4 leads ThT as a mainstream fluorescent tool for NAC indication nowadays (16)(17)(18), the extra supply of K + or Na + solution environment is required for G4 stably forming, as well as the large quantities of G4 in genome cannot be specifically image and distinguished by ThT. ...
... Therefore, considering the ThT fluorescence intensity are not only determined by the binding affinity, but also the factor of NAC's topological structure (13), as well as inspired by the mutation and bubbling (27) that might occur during DNA processing, and the widely accepted ThT 'light-switch' mechanism (15), we explored the A-C/A-G/A-G (CGG-AAA) cavity hairpin ThT-light nucleic acid switches (CHTLNAS) based on the combination of computer prediction and actual sequence verification. which has a stronger sequence programmability of the target section and a more conventional duplex conformation less subject to the restrictions imposed by formational conditions, serv-ing as a universal tool for effective fluorescent excitation and extending the application in the sensing and cellular imaging at the nucleic acid level on ThT. ...
Thioflavin T (ThT) is a classical fluorescent dye gaining prominence in current research regarding nucleic acid conformations (NACs). However, most NACs with the ability to excite ThT fluorescent are unique or form in demanding conditions, limiting the extensiveness and depth of ThT application in sensing and imaging. Therefore, this study proposed CGG-AAA mismatched cavity hairpin ThT-light nucleic acid switches (CHTLNAS) with excellent fluorescence excitation over 500-fold higher than spontaneous, 17∼20-fold higher than ssDNA and 2.5∼5-fold higher than complementary duplex. Based on the excellent fluorescence excitation, convenient conformation formation, good sequence programmability, and flexible allosteric ability (known as the Worm-crack pod mechanism mediated by the target), it achieved the label- and enzyme-free detection of tetracycline (TET) and berberine (BB) at the pM level within 10 min. Moreover, it was found enable to realize the sensitive tracking of intracellular carriers at the nM level of ThT entry concentration, and prolongated its cell nuclear-entry time of ThT over 8 h, overcoming the non-specific high background signal interference of ThT in the nuclear region, and expanding the diversified application of ThT in cell biology research. Therefore, CHTLNAS is a more universal, practical tool than G-quadruplex or other kinds of NACs for ThT development and utilization in sensing and imaging platforms.
... ThT acts as a molecular rotor upon excitation, i.e., photon absorption results in the mutual rotation of two ThT fragments -benzthiazole ring and benzene ring, see Fig. 1 (benzthiazole ring is marked with blue color, benzene ring with yellow color) [18,19]. Calculations demonstrate that in the ground state the angle between these two fragments, Φ, is 37°. ...
... Fmoc-FF is one of the most studied peptides for hydrogel preparation that alone or in combination with other substances can be used as a 3D scaffold for cartilage and bone tissue engineering [25]. Fig. 1 The structure of ThT and Fmoc-FF (left) and schematic representation of the phophysical processes in ThT according to Ref. [18]: upon excitation into the local excited (LE) state, relative rotation of two fragments in a free ThT molecule (marked with blue and yellow rectangles in the ThT structure) occurs, leading to the emergence of the twisted internal charge transfer (TICT) state. The TICT state exhibits ultrafast relaxation to the ground state on a ~1 ps scale. ...
... Molecules belonging to the FMR class are characterized by significant structural-conformational changes in the excited state, which manifests itself in significant dependence of the fluorescence intensity on temperature and viscosity of the microenvironment [9][10][11]. Such behavior is characteristic of thioflavin T [12][13][14], diphenylmethane (for example, auramine O [15][16][17]) and triphenylmethane [18,19] dyes, [p-(dialkylamino) benzylidene] malononitrile [20,21], a series of probes based on BODIPY [22][23][24]. In this regard, a number of new molecular rotors based on BODIPY [22,24], porphyrin dimers [25], and polymers with a stilbene-like chromophore group are tested as thermosensitive sensor molecules [26]. ...
... Previously, in a series of articles [13,14,27,28], it was shown that the thioflavin T (ThT) benzothiazole-aniline dye ( Fig. 1) belongs to the FMR class, and the quantum yield of its fluorescence is determined mainly by viscosity or the rigidity of the microenvironment. On the basis of quantum chemical calculations [12,14,29,30] a model of photophysical properties of ThT was proposed, where it is suggested that intramolecular charge transfer (TICT) occurs during photoexcitation, accompanied by a change in the dihedral angle between the aromatic fragments of the molecule from ϕ ∼ 37 to 90 • , leading to formation of a nonfluorescent TICT state. ...
Possible use of Thioflavin T based fluorescent molecular rotors as temperature sensors was assessed in this work. Fluorescent properties of Thioflavin T and its derivative 6-Me-BTA-2C in 99% glycerol were studied using steady-state and time-resolved fluorescence spectroscopy methods. For Thioflavin T in glycerol it has been found that temperature growth from 261 K to 353 K results in ~ 3 orders of magnitude increase of non-radiative decay rate constant k nr for the excited state of the molecule. Fluorescence decay studies by time-correlated single photon counting method showed that fluorescence decay kinetics of Thioflavin T in glycerol can be used for temperature measurements in the range 260-290 K. For 6-Me-BTA-2C molecule the range of the maximal sensitivity was shifted to higher temperatures (280-320 K). The obtained results about fluorescent properties and decay kinetics of Thioflavin T based dyes in highly viscous media can be used to develop nanoscale temperature sensors. Keywords: temperature sensor, fluorescent molecular rotor, nanothermometry, thioflavin T, TICT.
... A number of fluorescent probes have been intentionally designed to change their emission intensity or spectrum based on pH, polarity, and their immediate environment [26]. Interestingly, while the spectral shift of ThT upon binding to amyloid fibrils is well characterized [27][28][29][30], less is known about the capacity of ThS to change its emission spectrum in the presence of amyloid aggregates. ...
Amyloids are misfolded proteins that aggregate into fibrillar structures, the accumulation of which is associated with the pathogenesis of many neurodegenerative diseases, such as Alzheimer’s disease (AD). Early, sensitive detection of these misfolded aggregates is of great interest to the field, as amyloid deposition begins well before the presentation of clinical symptoms. Thioflavin-S (ThS) is a fluorescent probe commonly used to detect amyloid pathology. Protocols for ThS staining vary, but they often use high staining concentrations followed by differentiation, which causes varying levels of non-specific staining and potentially leaves more subtle amyloid deposition unidentified. In this study, we developed an optimized ThS staining protocol for the sensitive detection of β-amyloids in the widely used 5xFAD Alzheimer’s mouse model. Controlled dye concentrations together with fluorescence spectroscopy and advanced analytical methods enabled not only the visualization of plaque pathology, but also the detection of subtle and widespread protein misfolding throughout the 5xFAD white matter and greater parenchyma. Together, these findings demonstrate the efficacy of a controlled ThS staining protocol and highlight the potential use of ThS for the detection of protein misfolding that precedes clinical manifestation of disease.
... The tri exponential TRF emission decay for ThT in Bulk water has already been reported 85 . In addition, multiexponential fluorescence emission intensity decays for ThT in neat non-aqueous solvents, binary mixtures, and in amyloid fibril aggregates were experimentally detected that had used measurement set-ups with a temporal resolution of few tens of picosecond 85,86 . The multi-exponential ThT emission decays observed in our TRF measurements, therefore, reflect the generic nature of ThT lifetime decays in complex media (Fig. S2), indicating that ThT follows a similar trend with time for long incubation duration to that shown by the steady-state fluorescence intensity presented in Fig. 1. ...
Aggregation of the human islets amyloid polypeptide, or hIAPP, is linked to β-cell death in type II diabetes mellitus (T2DM). Different pancreatic β-cell environmental variables such as pH, insulin and metal ions play a key role in controlling the hIAPP aggregation. Since insulin and hIAPP are co-secreted, it is known from numerous studies that insulin suppresses hIAPP fibrillation by preventing the initial dimerization process. On the other hand, zinc and copper each have an inhibitory impact on hIAPP fibrillation, but copper promotes the production of toxic oligomers. Interestingly, the insulin oligomeric equilibrium is controlled by the concentration of zinc ions when the effect of insulin and zinc has been tested together. Lower zinc concentrations cause the equilibrium to shift towards the monomer and dimer states of insulin, which bind to monomeric hIAPP and stop it from developing into a fibril. On the other hand, the combined effects of copper and insulin have not yet been done. In this study, we have demonstrated how the presence of copper affects hIAPP aggregation and the toxicity of the resultant conformers with or without insulin. For this purpose, we have used a set of biophysical techniques, including NMR, fluorescence, CD etc., in combination with AFM and cell cytotoxicity assay. In the presence and/or absence of insulin, copper induces hIAPP to form structurally distinct stable toxic oligomers, deterring the fibrillation process. More specifically, the oligomers generated in the presence of insulin have slightly higher toxicity than those formed in the absence of insulin. This research will increase our understanding of the combined modulatory effect of two β-cell environmental factors on hIAPP aggregation.
... DCM-OH-2s compounds showed much lower x values comparing to Thioflavin T (ThT) (Figure 4a and 4b), a well-known RBF for well-aggregated amyloid fibrillar structures. 46 ...
Chromophores with zwitterionic excited-state intramolecular proton transfer (ESIPT) have been shown to have larger Stock shifts and red-shifted emission wavelengths compared to the conventional π-delocalized ESIPT molecules. However, there is still a dearth of design strategies to expand the current library of zwitterionic ESIPT compounds. Herein, we report a novel zwitterionic excited-state intramolecular proton transfer system enabled by addition of triazamacrocycle (TACN) fragments on a dicyanomethylene-4H-pyran (DCM) scaffold. The solvent-dependent steady-state photophysical studies and pKa measurements strongly support that the ESIPT process is more efficient with two TACN groups attached to the DCM scaffold and not affected by polar protic solvents. Impressively, compound DCM-OH-2-DT emits with a near-infrared (NIR) emission wavelength at 740 nm along with an uncommonly large Stokes shift of ~ 280 nm. Moreover, DCM-OH-2-DT shows high affinity towards soluble amyloid β (Aβ) oligomers in vitro and in 5xFAD mouse brain sections, and we have successfully applied DCM-OH-2-DT for the NIR fluorescence in vivo imaging of Aβ aggregates and demonstrated its potential use as an early diagnostic agent for AD. Overall, this study can provide a general molecular design strategy for developing new zwitterionic ESIPT compounds with NIR emission for further in vivo imaging applications.
... For quantitative analysis with fl uorescent markers an important factor is the ratio of the intensity (quantum yield) of fl uorescence for the incorporated dye and the free (uncombined) dye. For ThT, the quantum yield of which in aqueous solution is extremely low (0.0003 [11,12]) this ratio is >10 3 . For thiofl avin derivatives with a larger system of π conjugation the ratio of the fl uorescence intensity of the inserted and free dye is ≤ 200-300 on account of the higher quantum yield of fl uorescence of the derivatives in solution in the unbonded state. ...
The absorption and fluorescence spectra of a new styryl derivative of thioflavin T 2-{(1E,3E)-4-[4-(dimethylamino)-2,6-dimethylphenyl]buta-1,3-dien-1-yl}-3-ethyl-1,3-benzothiazol-3-ium tosylate (Th-C23) in solvents with different polarity and viscosity and also incorporated in the structure of amyloid fibrils and bovine serum albumin were investigated. A characteristic feature of the dye is an extremely low quantum yield of fluorescence in low-viscosity solvents (10–4 in water) which, however, increases significantly in viscous solutions and when it is incorporated in the structure of proteins or amyloid fibrils. In the latter case the quantum yield increases by 8∙103 times. On the basis of the experimental studies and quantum chemical calculations it was shown that Th-C23 exhibits the properties of a molecular rotor. The increase of the fluorescence quantum yield in viscous solutions and in the biopolymers results from limitation of the torsional rotation of the molecular fragments, leading to fluorescence quenching. The long-wavelength location of the absorption spectrum and the fluorescence spectrum of the new dye in the red region of the spectrum (520 and 600 nm) makes it possible to use it as a fluorescent marker that is sensitive to the viscosity (hardness) of the microenvironment not only in vitro but also in vivo.
... ThT is the molecular rotor molecule, whose fluorescence decay is determined by the viscosity of microenvironment. 32 The evolution of the ThT fluorescence lifetime for Fmoc-FF in a glycerol system is shown in Figure 3B(II), where it can be seen that the ThT lifetime for the lag phase that corresponds to the presence of spherical particle is τ ∼ 2500 ps, and the fibrillar hydrogel is characterized by the τ ∼ 1730 ps, whereas the distribution for the growth phase where the transition from spheres-to-fibers occurred is bimodal, indicating incorporation of the ThT fluorescence probe into the structures with different microenvironments. No multimodal distribution is observed for the growth phase in Fmoc-FF in water, demonstrating that such system is more homogeneous (see Figure 3B(I)). ...
Defrost sensors are a crucial element for proper functioning of the pharmaceutical cold chain. In this paper, the self-assembled peptide-based hydrogels were used to construct a sensitive defrost sensor for the transportation and storage of medications and biomaterials. The turbidity of the peptide hydrogel was employed as a marker of the temperature regime. The gelation kinetics under different conditions was studied to detect various stages of hydrogel structural transitions aimed at tuning the system properties. The developed sensor can be stored at room temperature for a long period, irreversibly indicates whether the product has been thawed, and can be adjusted to a specific temperature range and detection time.
... 472,503 The ThT conformation can play a critical role in fluorescence quantum yield, but that information can only be obtained by quantumchemical calculations, which is beyond the scope of this thesis. 479,480,[512][513][514] In summary, we observed the deterministic interaction of ThT on −solenoid adopting amyloids facilitating enhanced fluorescence and hence an accurate measurement of aggregation kinetic parameters. Amyloid surfaces with aromatic residues, hydrophobicity, and acidic residues improve accessibility for ThT binding, where basic residues hinder ThT binding. ...
Project 1- Structural dissection of amyloid fibrils of different TDP-43 constructs by solid-state NMRThe TAR DNA binding protein of 43 kDa (TDP-43) is observed as the main component in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar dementia (FTLD) cytoplasmic inclusions. TDP-43 consists of a well-folded N-terminal domain (NTD), two RNA recognition motif domains (RRM1 and RRM2) and an intrinsically disordered C-terminal domain (CTD). The prion-like C-terminal domain possesses most of the pathologically relevant mutations and plays a critical role in the spontaneous aggregation of TDP-43 and associated proteinopathy. We used a combination of electron microscopy, X‐ray fiber diffraction, Fourier‐transform infrared spectroscopy analysis, and solid‐state NMR spectroscopy to investigate the molecular organization of different TDP construct namely full-length TDP-43, two C-terminal fragments (TDP-35 and TDP-16) and a C-terminal truncated fragment (TDP-43 ∆GaroS2) in their fibrillar state. Although the different protein constructs exhibit similar fibril morphology and a typical cross-β signature by X-ray diffraction, solid-state NMR indicates that TDP-43 and TDP-35 share the same polymorphic molecular structure. At the same time, TDP-16 encompasses a well-ordered amyloid core. Our findings demonstrate that C-terminal fragments can adopt a different molecular conformation in isolation or the context of the full-length assembly, suggesting that the N-terminal and RRM domains play a critical role in the TDP-43 amyloid transition. ss-NMR studies of TDP-43 isoforms (sTDP43-1, sTDP43-2) responsible for ALS subtypes demonstrate that these isoforms can form amyloids without the CTD and adopt an amyloid fold similar to full-length TDP-43, suggesting a misfolding mechanism of NTD and RRM domains responsible for the amyloid transition in TDP43 isoforms.Project 2- Molecular mechanism of heterotypic amyloid signaling proteins NWD2/HET-s involved in Programmed cell death in fungiThe [Het-s] prion activates HET-S pore-forming protein and triggers programmed cell death (PCD) in the fungus Podospora anserina. The HET-s -solenoid fold act as a template for the amyloid transition of the HET-S prion-forming domain (PFD). A Nod-like receptor (NLR) protein called NWD2, encoded by the gene next to het-S possesses an N-terminal motif (R0) homologous to the elementary repeat unit of HET-s b-solenoid fold (R1, R2). The NLRs are associated with host defense and programmed cell death in plants, animals, and fungi. Upon activation, the N-terminal motif (R0) can adopt the b-solenoid fold, template the amyloid transition of the HET-S prion-forming domain, and trigger the downstream cell death pathway. In this work, we deduced the atomic resolution structure of the b-solenoid fold adopted by the functional amyloid NWD2 (R0) using solid-state NMR. Also, we showed that the templating mechanism involving NWD2 and HET-S achieved by the formation of a heterotypic amyloid interface via R0/R2, followed by PCD. Ss-NMR restraints determined an atomic-resolution structure of the heterotypic amyloid interface R0/R2.
... Molecular rotors such as thio-avin T are sensitive to viscosity and can be used to probe LLPS while also serving as a tool for identifying and visualizing protein aggregation and brilization. 152 Recently, a novel aggregation-induced emission uorogen, sodium 1,2-bis[4-(3sulfonatopropoxyl)phenyl]-1,2-diphenylethene (BSPOTPE), was demonstrated to be capable of reporting on LLPS by partitioning into droplets in vitro and uorescing in a viscositydependent manner. 153 The continued development of chemical tools capable of reporting on LLPS without the need for toxic or otherwise disruptive concentrations of 1,6-hexanediol will allow for less perturbative in vitro and in-cell assays. ...
Biomolecular phase transitions play an important role in organizing cellular processes in space and time. Methods and tools for studying these transitions, and the intrinsically disordered proteins (IDPs) that often drive them, are typically less developed than tools for studying their folded protein counterparts. In this perspective, we assess the current landscape of chemical tools for studying IDPs, with a specific focus on protein liquid-liquid phase separation (LLPS). We highlight methodologies that enable imaging and spectroscopic studies of these systems, including site-specific labeling with small molecules and the diverse range of capabilities offered by inteins and protein semisynthesis. We discuss strategies for introducing post-translational modifications that are central to IDP and LLPS function and regulation. We also investigate the nascent field of noncovalent small-molecule modulators of LLPS. We hope that this review of the state-of-the-art in chemical tools for interrogating IDPs and LLPS, along with an associated perspective on areas of unmet need, can serve as a valuable and timely resource for these rapidly expanding fields of study.
... Upon binding to β-sheet-rich structures, ThT gives a strong fluorescence signal at 482 nm when excited at 450 nm. The mechanism by which ThT fluorescence is enhanced upon binding to amyloids has been ascribed to the rotational immobilization of the C−C bond between the benzothiazole and aniline rings 26,27 (Figure 1b), which results in a dramatic shift in the ThT excitation maximum from 350 to 450 nm as shown in Figure 1c. Although ThT is an efficient reporter of fibril formation, its poor photophysical and binding properties make it ill-suited for detection of the small oligomeric species 28 that are critical precursors to fibril formation. ...
We report the effects of quercetin, a flavonoid present in the human diet, on early stage beta-amyloid (Aβ) aggregation, a seminal event in Alzheimer's disease. Molecular level changes in Aβ arrangements are monitored by time-resolved emission spectral (TRES) measurements of the fluorescence of Aβ's single tyrosine intrinsic fluorophore (Tyr). The results suggest that quercetin binds β-amyloid oligomers at early stages of their aggregation, which leads to the formation of modified oligomers and hinders the creation of β-sheet structures, potentially preventing the onset of Alzheimer's disease.
... It was already shown that at the amyloidogenic conditions causing partial unfolding of insulin, at least two major populations of partially unfolded intermediates are present, in which all three disulfide bonds remain intact (Ahmad et al., 2005;Zako, et al., 2009;Kurouski et al., 2012). The differences between these intermediates cause various fibrillization pathways that might generate morphologically different fibrils, which represent the basis of the amyloid fibril polymorphisms and the morphology of fibrils subsequently affects also the ThT fluorescence (Dzwolak et al., 2004;Stsiapura et al., 2008;Chatani et al., 2015;Sneideris et al., 2015;Eisenberg, and Sawaya, 2017). We demonstrated that the propensity of insulin to form fibrils is affected by Chobimalt in a concentration-and time-dependent manner (Figure 2, 3). ...
The amyloidogenic self-assembly of many peptides and proteins largely depends on external conditions. Among amyloid-prone proteins, insulin attracts attention because of its physiological and therapeutic importance. In the present work, the amyloid aggregation of insulin is studied in the presence of cholesterol-based detergent, Chobimalt. The strategy to elucidate the Chobimalt-induced effect on insulin fibrillogenesis is based on performing the concentration- and time-dependent analysis using a combination of different experimental techniques, such as ThT fluorescence assay, CD, AFM, SANS, and SAXS. While at the lowest Chobimalt concentration (0.1 µM; insulin to Chobimalt molar ratio of 1:0.004) the formation of insulin fibrils was not affected, the gradual increase of Chobimalt concentration (up to 100 µM; molar ratio of 1:4) led to a significant increase in ThT fluorescence, and the maximal ThT fluorescence was 3-4-fold higher than the control insulin fibril’s ThT fluorescence intensity. Kinetic studies confirm the dose-dependent experimental results. Depending on the concentration of Chobimalt, either (i) no effect is observed, or (ii) significantly, ∼10-times prolonged lag-phases accompanied by the substantial, ∼ 3-fold higher relative ThT fluorescence intensities at the steady-state phase are recorded. In addition, at certain concentrations of Chobimalt, changes in the elongation-phase are noticed. An increase in the Chobimalt concentrations also triggers the formation of insulin fibrils with sharply altered morphological appearance. The fibrils appear to be more flexible and wavy-like with a tendency to form circles. SANS and SAXS data also revealed the morphology changes of amyloid fibrils in the presence of Chobimalt. Amyloid aggregation requires the formation of unfolded intermediates, which subsequently generate amyloidogenic nuclei. We hypothesize that the different morphology of the formed insulin fibrils is the result of the gradual binding of Chobimalt to different binding sites on unfolded insulin. A similar explanation and the existence of such binding sites with different binding energies was shown previously for the nonionic detergent. Thus, the data also emphasize the importance of a protein partially-unfolded state which undergoes the process of fibrils formation; i.e., certain experimental conditions or the presence of additives may dramatically change not only kinetics but also the morphology of fibrillar aggregates.
... This has a specific sensitivity to environmental polarity, presence of specific residues (e.g., aromatics), or spacing between β-strands 40 . In aqueous environment ThT has a lifetime in the picosecond range, while longer lifetimes are measured in media with higher viscosity 41 . FLIM measurements were analyzed using the phasor approach, detailed in the Methods section, that provides a global view of the decay of fluorescent molecules, not imposing any specific model as required by fitting procedures (Fig. 3) 42,43 . ...
Some marine organisms can resist to aqueous tidal environments and adhere tightly on wet surface. This behavior has raised increasing attention for potential applications in medicine, biomaterials, and tissue engineering. In mussels, adhesive forces to the rock are the resultant of proteinic fibrous formations called byssus. We present the solution structure of Pvfp-5β, one of the three byssal plaque proteins secreted by the Asian green mussel Perna viridis, and the component responsible for initiating interactions with the substrate. We demonstrate that Pvfp-5β has a stably folded structure in agreement with the presence in the sequence of two EGF motifs. The structure is highly rigid except for a few residues affected by slow local motions in the µs-ms time scale, and differs from the model calculated by artificial intelligence methods for the relative orientation of the EGF modules, which is something where computational methods still underperform. We also show that Pvfp-5β is able to coacervate even with no DOPA modification, giving thus insights both for understanding the adhesion mechanism of adhesive mussel proteins, and developing of biomaterials. Solution structure of byssal plaque protein Pvfp-5β secreted by the Asian green mussel Perna viridis gives molecular insight into mussel adhesion on wet surfaces.
... With our Gelation was accompanied with some textural changes. Several authors claimed an increase in ThT fluorescence of protein-free dispersions when increasing the viscosity of the medium (Kuzmitskii & Stepuro, 2017;Stsiapura et al., 2008;Sulatskaya, Sulatsky, Antifeeva, Kuznetsova, & Turoverov, 2019). ThT fluorescence is based on deactivation of the excited state after excitation at 440 nm. ...
Heating aqueous solutions of ovalbumin (OVA) may cause gel formation. When heated at pH conditions close to the protein's isoelectric point (towards neutral pH), turbid particulate gels are formed, whereas at acidic pH fine-stranded, transparent gels are formed already at lower concentrations. Here, transparent gels were formed when subjecting 2.0% OVA to a combined heat (78 °C for 22 h at pH 7) and trypsin (37 °C for 48 h) treatment. Transmission electron microscopy clearly revealed the presence of long, straight OVA fibrils which contributed to the gel formation. Quartz crystal microbalance with dissipation (adsorption of small structures), size exclusion – HPLC (presence of both structures larger and smaller than native OVA) and atomic force microscopy (presence of long fibrils with a higher thickness, whereas heated OVA mainly showed amorphous aggregates) analyses confirmed that the additional enzymatic treatment was able to break down the amorphous aggregates formed by heating OVA into peptides, which then partly re-assembled into longer OVA fibrils. The above mentioned heat and enzymatic treatment conditions brought about gelation after 17 h with a gel strength of 68 Pa which broke at a stress of 38 Pa. By varying the temperature during heat (58–88 °C) and enzymatic (27–67 °C) treatments, gels were formed the fastest when heated at 78 °C followed by enzymatic treatment at 57 °C. A design of experiments for evaluating the impact of OVA and trypsin concentration revealed that the fastest gelation occurred at the higher considered OVA and trypsin concentrations. Additionally, the gel strength was also higher under the latter conditions. It is clear that different gel characteristics can be reached when varying the different process conditions, creating the opportunity for reconsidering the formulation of various foods such as jellies, marmalades and desserts.
The viscosity and crowding of biological environment are considered vital for the correct cellular function, and alterations in these parameters are known to underly a number of pathologies including diabetes, malaria, cancer and neurodegenerative diseases, to name a few. Over the last decades, fluorescent molecular probes termed molecular rotors proved extremely useful for exploring viscosity, crowding, and underlying molecular interactions in biologically relevant settings. In this review, we will discuss the basic principles underpinning the functionality of these probes and will review advances in their use as sensors for lipid order, protein crowding and conformation, temperature and non‐canonical nucleic acid structures in live cells and other relevant biological settings.
The viscosity and crowding of biological environment are considered vital for the correct cellular function, and alterations in these parameters are known to underly a number of pathologies including diabetes, malaria, cancer and neurodegenerative diseases, to name a few. Over the last decades, fluorescent molecular probes termed molecular rotors proved extremely useful for exploring viscosity, crowding, and underlying molecular interactions in biologically relevant settings. In this review, we will discuss the basic principles underpinning the functionality of these probes and will review advances in their use as sensors for lipid order, protein crowding and conformation, temperature and non‐canonical nucleic acid structures in live cells and other relevant biological settings.
Measuring the diffusivity of molecules is the first step towards understanding their dependence and controlling diffusion, but the challenge increases with the decrease of molecular size, particularly for non‐fluorescent and non‐reactive molecules such as solvents. Here, we demonstrate the capability to monitor the solvent exchange process within the micropores of silica with millisecond time resolution, by simply embedding a rotor‐based fluorophore (thioflavin T) in colloidal silica nanoparticles. Basically, the silica provides an extreme case of viscous microenvironment, which is affected by the polarity of the solvents. The fluorescence intensity traces could be well fitted to the Fickian diffusion model, allowing analytical solution of the diffusion process and revealing the diffusion coefficients. The validation experiments, involving the water‐to‐ethanol and ethanol‐to‐water solvent exchange, the comparison of different drying conditions, and the variation in the degree of cross‐linking in silica, confirmed the effectiveness and sensitivity of this method for characterizing diffusion in silica micropores. This work focuses on the method development of measuring diffusivity and the high temporal resolution in tracking solvent exchange dynamics over a short distance (within 165 nm) opens enormous possibilities for further studies.
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Insulin forms liner fibrils and aggregates through a physical phenomenon where partially unfolded polypeptide molecules interact to oligomerize through various noncovalent interactions. Expectedly, this manifestation reduces (bio)availability of active insulin...
Chromophores with zwitterionic excited-state intramolecular proton transfer (ESIPT) have been shown to have larger Stock shifts and red-shifted emission wavelengths compared to the conventional π-delocalized ESIPT molecules. However, there is still a dearth of design strategies to expand the current library of zwitterionic ESIPT compounds. Herein, we report a novel zwitterionic excited-state intramolecular proton transfer system enabled by addition of 1,4,7-triazacyclononane (TACN) fragments on a dicyanomethylene-4H-pyran (DCM) scaffold. The solvent-dependent steady-state photophysical studies, pKa measurements, and computational analysis strongly support that the ESIPT process is more efficient with two TACN groups attached to the DCM scaffold and not affected by polar protic solvents. Impressively, compound DCM-OH-2-DT exhibits a near-infrared (NIR) emission at 740 nm along with an uncommonly large Stokes shift. Moreover, DCM-OH-2-DT shows high affinity towards soluble amyloid β (Aβ) oligomers in vitro and in 5xFAD mouse brain sections, and we have successfully applied DCM-OH-2-DT for the in vivo imaging of Aβ aggregates and demonstrated its potential use as an early diagnostic agent for AD. Overall, this study can provide a general molecular design strategy for developing new zwitterionic ESIPT compounds with NIR emission in vivo imaging applications.
Non-canonical secondary structures in DNA are increasingly being revealed as critical players in DNA metabolism, including modulating the accessibility and activity of promoters. These structures comprise the so-called G-quadruplexes (G4s) that are formed from sequences rich in guanine bases. Using a well-defined transcriptional reporter system, we sought to systematically investigate the impact of the presence of G4 structures on transcription in yeast S. cerevisiae. To this aim, different G4 prone sequences were modeled to vary the chance of intramolecular G4 formation, analyzed in vitro by Thioflavin T binding test and circular dichroism and then placed at the yeast ADE2 locus on chromosome XV, downstream and adjacent to a P53 response element (RE) and upstream from a minimal CYC1 promoter and Luciferase 1 (LUC1) reporter gene in isogenic strains. While the minimal CYC1 promoter provides for basal reporter activity, the P53 RE enables LUC1 transactivation under the control of the human P53 family proteins expressed under the inducible GAL1 promoter. Thus, the impact of the different G4 prone sequences on both basal and P53 family proteins dependent expression was measured after shifting the yeast cells onto galactose containing medium. The results showed that the presence of G4 prone sequences upstream of a yeast minimal promoter can increase its basal activity proportionally to their potential to form intramolecular G4 structures; consequently, this improved accessibility, when present near the target binding site of P53 family transcription factors can be exploited in order to regulate the transcriptional activity of P53, P63 and P73 proteins.
Thioflavin t (THT) is a well-known molecular rotor extensively used to detect amyloid-like structures. But THT shows very weak emission in water. In this article, we have found that THT shows very strong emission in the presence of cellulose nanocrystals (CNCs). Steady-state and time-resolved emission techniques have been used to study the strong emission of THT in aqueous CNC dispersion. The time-resolved study showed that in the presence of CNCs, the lifetime increased by ∼1500 fold compared to pure water (<1 ps). To know the nature of interaction and also the reason for this increase in emission zeta potential, stimuli-dependent and temperature-dependent studies have been carried out. These studies proposed that electrostatic interaction is the main factor for this binding of THT with CNCs. Further, the addition of another anionic lipophilic dye, merocyanine 540 (MC540), with CNCs-THT in both BSA protein (CIE: 0.33, 0.32) and TX-100 micellar (4.5 mM) (CIE: 0.32, 0.30) solutions produced excellent white light emission. Lifetime decay and absorption studies proposed a possible fluorescence resonance energy transfer mechanism in this generation of white light emission.
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Cytochrome c oxidase (CcO) is the terminal enzyme in the respiratory electron transport chain of aerobic organisms. It is a redox-driven proton pump that converts atmospheric oxygen to water and couples the oxygen reduction, generating a membrane proton gradient that subsequently drives ATP synthesis. The function of CcO as a biomolecular nanomachine that transforms the energy of redox reaction into protonmotive force across a biological membrane has been the subject of intense research, debate, and controversy. For a long time, the molecular mechanism of electron transfer coupled to proton pumping in CcO has been one of the central unsolved problems in biochemistry, molecular biology, and bioenergetics. The enzyme structure has been solved for several organisms; however, details of its molecular mechanism of proton pumping still remain elusive. Mainly, the nature and position of the proton-loading site, a key element of the mechanism, are under dispute. However, nowadays, many essential details and principles have emerged with the accelerating progress in this field. Recent calculations indicate that one of the histidine ligands of the CuB center, His291, may play the role of the pumping element. In this chapter, we review the first principles calculation used to study models of the catalytic center of CcO and calculate the pKa of the His291 residue for both the reduced and oxidized states of the enzyme catalytic center. The combined density functional theory (DFT) and continuum electrostatic calculations (QM/CE method) are employed to explore the coupling of the conformational changes of Glu242 residue, the primary proton donor of both chemical and pump protons, to its pKa, and the pKa of His291, a putative proton-loading site of the pumping model. The pKa values of His291 and Glu242, the two crucial residues of the model, are calculated for different redox states of the enzyme, and the influence of various factors on the pKas is analyzed in detail. To understand how different factors affect the apparent pKa values of His291 and Glu242, we have considered several computational QM/CE models of the membrane‒enzyme‒cavities‒solvent system and tested different dielectric properties of the water-filled cavities. In addition, the DFT is applied to two different sizes of quantum-mechanical (QM) systems of interest. We also review the structure and function of CcO, describe the proposed mechanism of proton pumping, identify the fundamental problems that can be addressed computationally in this area and describe the methods for their solution. The outstanding theoretical and computational challenges of the area are also discussed.
© 2023 Nova Sience Publishers, Inc.
Keywords: bioenergetics, heme-copper oxidases, nitric-oxide reductase, copper center, heme, binuclear complex, histidine ligand, bovine, proton pumping, molecular mechanism, redox-coupled pKa, pKa calculations, DFT, reaction and protein field.
Categories: 2022, Advances in Biology (Numbered Series) *** New in 2022 ***, Biology, Biology Books, Life Sciences, Newly Published Books, Nova, Science and Technology
Imaging amyloid-beta (Aβ) aggregation is critical for understanding the pathology and aiding the pre-symptomatic intervention of Alzheimer's disease (AD). Amyloid aggregation consists of multiple phases with increasing viscosities and demands probes with broad dynamic ranges and gradient sensitivities for continuous monitoring. Yet, existing probes designed based on the twisted intramolecular charge transfer (TICT) mechanism mainly focused on donor engineering, limiting the sensitivities and/or dynamic ranges of these fluorophores to a narrow window. Herein, using quantum chemical calculations, we investigated multiple factors affecting the TICT process of fluorophores. It includes the conjugation length, the net charge of the fluorophore scaffold, the donor strength, and the geometric pre-twisting. We have established an integrative framework for tuning TICT tendencies. Based on this framework, a platter of hemicyanines with varied sensitivities and dynamic ranges is synthesized, forming a sensor array and enabling the observation of various stages of Aβ aggregations. This approach will significantly facilitate the development of TICT-based fluorescent probes with tailored environmental sensitivities for numerous applications.
The absorption and fluorescence spectra of a new styryl derivative of thioflavin T tosylate 2-{(1E,3E)-4-[4-(dimethylamino)-2,6-dimethylphenyl]buta-1,3-dien-1-yl}- 3-ethyl-1,3-benzothiazolium-3 (Th-C23) in solvents of different polarity and viscosity, as well as when it is incorporated into the structure of amyloid fibrils and bovine serum albumin are investigated. A characteristic feature of the dye is an extremely low fluorescence quantum yield in low-viscosity solvents (10–4 in water), which, however, increases significantly in viscous solutions and when incorporated into the structure of proteins or amyloid fibrils. In the latter case, the quantum yield increases by 8х103 times. Based on experimental studies and quantum chemical calculations, it is shown that Th-C23 exhibits molecular rotor properties. An increase in the fluorescence quantum yield in viscous solutions and upon incorporation into biopolymers is the result of limiting the torsion rotation of molecular fragments, leading to fluorescence quenching. The long-wavelength position of the absorption spectrum and the fluorescence spectrum of the new dye in the red region of the spectrum (520 and 600 nm) makes it possible to use it as a fluorescent marker sensitive to the viscosity (hardness) of the microenvironment not only in vitro, but also in vivo.
Aggregated protein is the common cause of a wide variety of neurodegenerative diseases, such as Alzheimer’s disease (AD), Parkinson’s disease, etc. It is proven that protein aggregation like amyloid β (Aβ) is one of the critical factors causing AD and, its diagnosis in the early stages of the disease is important for the treatment or prevention of AD. To have a better understanding of protein aggregation and its pathologies, there is a huge need to design and develop new and more trustworthy probe molecules for in vitro amyloid quantification and in vivo amyloid imaging. In this study, 17 new biomarker compounds, have been synthesized from benzofuranone derivatives, to detect and identify amyloid in vitro (dye-binding assay) as well as in the cell by staining method. According to the results, some of these synthetic derivatives can be considered suitable identifiers and quantifiers to detect amyloid fibrils in vitro. Compared to thioflavin T, 4 probes out of 17 probes have shown good results in selectivity and detectability of Aβ depositions, and their binding properties were also confirmed with in silico analysis. The drug-likeness prediction results for selected compounds by the Swiss ADME server show a satisfactory percentage of blood-brain barrier (BBB) permeability and gastrointestinal (GI) absorption. Among all of them, compound 10 was able to show better binding properties than others, and in vivo study showed that this compound was capable of detecting intracellular amyloid.
Communicated by Ramaswamy H. Sarma
Amyloid aggregation of protein is linked to many neurodegenerative diseases. Identification of small molecules capable of targeting amyloidogenic proteins has gained significant importance. Introduction of hydrophobic and hydrogen bonding interactions through site-specific binding of small molecular ligand to protein can effectively modulate the protein aggregation pathway. Here, we investigate the possible roles of three different bile acids, cholic acid (CA), taurocholic acid (TCA), and lithocholic acid (LCA) with varying hydrophobic and hydrogen bonding properties in inhibiting protein fibrillation. Bile acids are an important class of steroid compounds that are synthesized in the liver from cholesterol. Increasing evidence suggests that altered taurine transport, cholesterol metabolism, and bile acid synthesis have strong implications in Alzheimer's disease. We find that the hydrophilic bile acids, CA and TCA (taurine conjugated form of CA), are substantially more efficient inhibitors of lysozyme fibrillation than the most hydrophobic secondary bile acid LCA. Although LCA binds more strongly with the protein and masks the Trp residues more prominently through hydrophobic interactions, the lesser extent of hydrogen bonding interactions at the active site has made LCA a relatively weaker inhibitor of HEWL aggregation than CA and TCA. The introduction of a greater number of hydrogen bonding channels by CA and TCA with several key amino acid residues which are prone to form oligomers and fibrils has weakened the protein's internal hydrogen bonding capabilities for undergoing amyloid aggregation.
The development of effective methods for the detection of protein misfolding is highly beneficial for early stage medical diagnosis and the prevention of many neurodegenerative diseases. Self-assembled plasmonic nanoantennas with precisely tunable nanogaps show extraordinary electromagnetic enhancement, generating extreme signal amplification imperative for the design of ultrasensitive biosensors for point of care applications. Herein, we report the custom arrangement of Au nanobipyramid (Au NBP) monomer and dimer nanoantennas engineered precisely based on the DNA origami technique. Furthermore, we demonstrate the SERS based detection of thioflavin T (ThT), a well-established marker for the detection of amyloid fibril formation, where G-Quadruplexes govern the site-specific attachment of ThT in the plasmonic hotspot. This is the first study for the SERS based detection of the ThT dye attached specifically using a G-Quadruplex complex. The spectroscopic signals of ThT were greatly enhanced due to the designed nanoantennas demonstrating their potential as superior SERS substrates. This study paves the way for boosting the design of next-generation diagnostic tools for the specific and precise detection of various target disease biomarkers using molecular probes.
Thioflavin T (ThT) is a typical fluorescent marker for detecting the formation of amyloid fibrils, because its fluorescence intensity increases by more than 2 orders of magnitude upon complexation with the fibrils. Strong electrostatic fields on protein surfaces are known to be a significant factor in chemical reactions and biological functions. Therefore, ThT bound to amyloid fibrils must experience strong electric fields. This study employed electroabsorption and Stark fluorescence spectroscopies to clarify the effects of external electric fields on the photophysics of ThT. The absorption spectrum shows two bands ascribed to locally excited (LE) and charge transfer (CT) states. Coupling between the LE and CT states is enhanced in the presence of an external electric field, resulting in fluorescence quenching. The electric field strength of the amyloid fibril surface was inferred from the fluorescence quenching efficiency of ThT.
A bifunctional TPE based fluorescent sensor (TPE-Q) for both liquid viscosity and Amyloidβ (Aβ) have been synthesized straight-forwardly by introduction of ethylene glycol chain and quaternary ammonium to classical tetraphenyl...
Human pancreatic lipase is a symbolic biomarker for the diagnosis of acute pancreatitis, which has profound significance for clinical detection and disease treatment. Herein, we first demonstrate a paper-based lipase sensor via a phase separation-induced viscosity change. Lipase catalyzes triolein to produce oleic acid and glycerol. Adding an excess of Ca2+ produces calcium oleate. The remaining Ca2+ binds with sodium alginate, triggering hydrogelation with an "egg-box" structure. The viscosity change of the aqueous solution induced by the phase separation process can be quantified by measuring the solution flow distance on a pH test paper. The paper-based lipase sensor has high sensitivity with a detection limit of 0.052 U/mL and also shows excellent specificity. Additionally, it is also utilized for quantitative lipase analysis in human serum samples to exhibit its potency in acute pancreatitis detection. This method overcomes the drawbacks of low sensitivity, slow response, and poor reproducibility caused by the nonuniform distribution of the highly viscous hydrogel on the sensing interface in existing approaches. In conclusion, thanks to the prominent characteristics of high portability, low cost, and easy operation, it is prospective for simple quantitative detection of lipase and has great potential for commercialization.
Precise tuning of the fluorescence quantum yield, vital for countless applications of fluorophores, remains exceptionally challenging due to numerous factors affecting energy dissipation phenomena often leading to its counterintuitive behavior. In contrast to the absorption and emission wavelength which can be precisely shifted to the desired range by simple structural changes, no general strategy exists for controllable modification of the fluorescence quantum yield. The rigidification of the molecular skeleton is known to usually enhance the emission and can be practically realized via the limiting molecular vibrations by aggregation. However, the subtle balance between the abundant possible radiative and non-radiative decay pathways makes the final picture exceptionally sophisticated. In the present study, a series of nine fluorophores obtained by peripheral substitution with two relatively mild electron donating and electron withdrawing groups are reported. The obtained fluorescence quantum yields range from dark to ultra-bright and the extreme values are obtained for the isomeric molecules. These severe changes in emission efficiency have been shown to arise from the complex relationship between the Franck-Condon excited state and conical intersection position. The experimental findings are rationalized by the advanced quantum chemical calculations delivering good correlation between the measured emission parameters and theoretical radiative and internal conversion rate constants. Therefore, the described substituent exchange provides a method to rigorously adjust the properties of molecular probes structurally similar to thioflavin T.
Fibrillar aggregates of the α-synuclein (αS) protein are the hallmark of Parkinson’s Disease and related neurodegenerative disorders. Characterization of the effects of mutations and post-translational modifications (PTMs) on the αS aggregation rate can provide insight into the mechanism of fibril formation, which remains elusive in spite of intense study. A comprehensive collection (375 examples) of mutant and PTM aggregation rate data measured using the fluorescent probe thioflavin T is presented, as well as a summary of the effects of fluorescent labeling on αS aggregation (20 examples). A curated set of 131 single mutant de novo aggregation experiments are normalized to wild type controls and analyzed in terms of structural data for the monomer and fibrillar forms of αS. These tabulated data serve as a resource to the community to help in interpretation of aggregation experiments and to potentially be used as inputs for computational models of aggregation.
X-ray crystallography has long been a key method in solving the three-dimensional structure of proteins. Structural information is essential for unraveling the molecular function of proteins and structure-based drug design. However, there are several obstacles associated with the structural determination of proteins using X-ray crystallography, such as the generation of a large amount of protein samples, instability of purified proteins, and difficulty in obtaining large and well-diffracting crystals, all of which can prolong the process of determining the crystal structure, from months to years. Over the past decade, new techniques and strategies have been developed to assist X-ray crystallographers in overcoming some of these obstacles. In this chapter, we discuss some of these technological advances. Familiarity with these new developments would benefit researchers in both academic and industrial environments who study macromolecular structural dynamics using X-ray crystallography.
Fluorescence is a phenomenon of broad appeal and several applications. Among the most important ones are fluorescence-based assays and methods that allow the analysis of biomolecular structure and dynamics. For structural studies, one can assess the relative orientation of protein fragments to the polarity of the environment and describe conformational changes. Moreover, both small ligand binding and oligomerization are easily studied with fluorescence-based techniques. Dissociation constant and other kinetics parameters can also be calculated from such data. Finally, biomolecules might also be identified and tracked in vivo in real time and at high resolution. The biomolecule of interest may be autofluorescent, or it may need external labeling; the availability of different fluorophores is also discussed. Here, we discuss essential applications of fluorescence-based methods providing an overview of the vast possibilities they offer for researchers.
Diversity in protein conformations is closely associated with their function. Methods exploring conformation alterations are valuable to harness the disease-relevant changes in protein structure for therapeutic purposes. Although an array of methods are available to analyze tertiary structures in proteins, the quantitative and qualitative analysis of protein conformations is comprehensively studied using multiple spectroscopic methods. Spectroscopic techniques involve the interaction of electromagnetic radiations with proteins in solution. They are discrete in terms of the regions of electromagnetic spectrum utilized for interaction and the properties of the biological sample, which are altered during excitation by radiation. Spectroscopic methods excel in providing information about conformational transitions in both the active soluble and inactive aggregate forms of proteins. This chapter provides an overview of the methodology of various spectroscopic techniques and discusses their application in understanding the structural properties of oligomeric proteins.
Small-angle scattering (SAS) techniques provide information regarding molecular, macromolecular, and supramolecular distances; interactions; shapes; composition; and so on. These techniques involve small-angle X-ray scattering, small-angle neutron scattering, and small-angle light scattering when using X-rays, neutrons, or visible light as probes, respectively. At a spatial range between 1 and 100 nm, the type of information that SAS techniques bring roughly overlaps with other techniques. The closest relatives are dynamic and static light scattering and microscopy techniques, but neither can provide information on biomolecular dynamics in the way that SAS techniques can. SAS techniques have been hyphenated to many other characterization techniques to obtain relevant information under nonequilibrium conditions. Aided by powerful modeling, these techniques have gained an indisputable place in biomolecule analysis and are becoming more widespread. This chapter aims to present the state-of-the-art SAS techniques for the inexperienced and SAS enthusiast users.
Chiral materials related to circularly polarized luminescence (CPL) are a rapidly developing new field that has broad application prospects in optoelectronic devices, selective recognition, biomedicine, and other fields. Biomolecules naturally chiral, and biofunctional chiral materials attracted increasing attention because of their unique biocompatibility, chiral recognition and coding. However, there is little discussion on the biomolecule‐based CPL till now. In this paper, the latest progress in CPL materials based on biomolecules is reviewed, including the chiral construction of bio‐based CPL materials and their emerging applications. In addition, we put forward the main challenges and future prospects of bio‐based CPL materials, hoping this work would provide new perspectives and insights for more related research.
Chiral materials related to circularly polarized luminescence (CPL) are a rapidly developing new field that has broad application prospects in optoelectronic devices, selective recognition, biomedicine, and other fields. Biomolecules naturally chiral, and biofunctional chiral materials attracted increasing attention because of their unique biocompatibility, chiral recognition and coding. However, there is little discussion on the biomolecule‐based CPL till now. In this paper, the latest progress in CPL materials based on biomolecules is reviewed, including the chiral construction of bio‐based CPL materials and their emerging applications. In addition, we put forward the main challenges and future prospects of bio‐based CPL materials, hoping this work would provide new perspectives and insights for more related research.
Targeting amyloidosis requires high-resolution insight into the underlying mechanisms of amyloid aggregation. The sequence-specific intrinsic properties of a peptide or protein largely govern the amyloidogenic propensity. Thus, it is essential to delineate the structural motifs that define the subsequent downstream amyloidogenic cascade of events. Additionally, it is important to understand the role played by extrinsic factors, such as temperature or sample agitation, in modulating the overall energy barrier that prompts divergent nucleation events. Consequently, these changes can affect the fibrillation kinetics, resulting in structurally and functionally distinct amyloidogenic conformers associated with disease pathogenesis. Here, we have focused on human Islet Polypeptide (hIAPP) amyloidogenesis for the full-length peptide along with its N- and C-terminal fragments, under different temperatures and sample agitation conditions. This helped us to gain a comprehensive understanding of the intrinsic role of specific functional epitopes in the primary structure of the peptide that regulates amyloidogenesis and subsequent cytotoxicity. Intriguingly, our study involving an array of biophysical experiments and ex vivo data suggests a direct influence of external changes on the C-terminal fibrillating sequence. Furthermore, the observations indicate a possible collaborative role of this segment in nucleating hIAPP amyloidogenesis in a physiological scenario, thus making it a potential target for future therapeutic interventions.
We report the application of two iodinated boron dipyrromethene (BODIPY) photocatalysts as theranostic compounds against pathogenic bacterial biofilms. These derivatives, which are activated through a polarity effect upon binding, have exquisite selectivity towards amyloids in the biofilm extracellular matrix (ECM). This property can be exploited in fluorescence microscopy to stain the ECM of uropathogenic bacterial biofilms, displaying a superior selectivity compared to Thioflavin T, the gold standard for amyloid detection. Moreover, we show that the ability of these compounds to photosensitize reactive oxygen species upon binding can be used to weaken the mechanical barrier of the ECM and restore the activity of conventional antimicrobial agents, whose effect is strongly limited in intact biofilms. The combination of activatable near-infrared fluorescence and photo-oxygenation properties of these compounds upon binding to amyloid structures provides a powerful theranostic tool for simultaneous detection and treatment of pathogenic biofilm infections.
At present, nanoplastics have been detected in food and the environment, but they have serious impacts on the human body. As one of the typical representatives of nanoplastics, polystyrene (PS) is generally used as an experimental object. Few studies found that PS could modulate the formation of amyloid fibrils, leading to the occurrence of diseases. However, its submicron-scale effects remain elusive. Thus, this study aimed to explore the interaction between PS of particle size 100-500 nm and hen egg-white lysozyme (HEWL). The results showed that PS of size 400 nm markedly promoted the primary nucleation step of amyloid fibril formation, and fibrils had more small fragments compared with PS of size 100 nm in the control and sample groups. PS of larger particle size changed the spatial structure of HEWL significantly. This study analyzed the experimental results from the perspective of protein corona and thermodynamics. The study confirmed that PS was able to form protein corona with HEWL in the initial stage, which was mainly driven by hydrophobic interactions. More importantly, the interface and junction of the protein corona were the main sites for the formation of amyloid fibrils. This study highlighted the role of submicron particle size and discussed the toxic effects of nanoparticles.
The structural changes during the intramolecular charge transfer (ICT) of nitroaromatic chromophores, 4-dimethylamino-4′-nitrobiphenyl (DNBP) and 4-dimethylamino-4′-nitrostilbene (DNS) were investigated by femtosecond stimulated Raman spectroscopy (FSRS) with both high spectral and temporal resolutions. The kinetically resolved Raman spectra of DNBP and DNS in the locally-excited and charge-transferred states of the S1 state appear distinct, especially in the skeletal vibrational modes of biphenyl and stilbene including 8a and C=C. The 8a of two phenyls and the C=C of the central ethylene group (only for stilbene), which are strongly coupled in the planar geometries, are broken with the twist of nitrophenyl group with the ICT. Time-resolved vibrational spectroscopy measurements and the time-dependent density functional theory simulations support the ultrafast ICT dynamics of 220-480 fs with the twist of nitrophenyl group occurring in the S1 state of the nitroaromatic chromophores. While the ICT of DNBP occurs via a barrier-less pathway, the ICT coordinates of DNS are strongly coupled to several low-frequency out-of-phase deformation modes relevant to the twist of the nitrophenyl group.
Cyclin - dependent kinase 6 (CDK6) is an important protein kinase that regulates cell growth, development, cell metabolism, inflammation, and apoptosis. Its overexpression is associated with reprogramming glucose metabolism through alternative pathways and apoptosis, which ultimately plays a significant role in cancer development. In the present study, we have investigated the structural and conformational changes in CDK6 at varying pH employing a multi-spectroscopic approach. Circular dichroism (CD) spectroscopy revealed at extremely acidic conditions (pH 2.0–4.0), the secondary structure of CDK6 got significantly disrupted, leading to aggregates formation. These aggregates were further characterized by employing Thioflavin T (ThT) fluorescence. No significant secondary structural changes were observed over the alkaline pH range (pH 7.0–11.0). Further, fluorescence and UV spectroscopy revealed that the tertiary structure of CDK6 was disrupted under extremely acidic conditions, with slight alteration occurring in mild acidic conditions. The tertiary structure remains intact over the entire alkaline range. Additionally, enzyme assay provided an insight into the functional aspect of CDK at varying pH; CDK6 activity was optimal in the pH range of 7.0–8.0. This study will provide a platform that provides newer insights into the pH-dependent dynamics and functional behavior of CDK6 in different CDK6 directed diseased conditions, viz. different types of cancers where changes in pH contribute to cancer development.
The fluorescence and absorption spectra of the dyestuff thioflavine T are strongly affected by the freezing of its solution in water. The spectral changes are thought to be caused by dimerization of dyestuff ions which can be interpreted in terms of the usual cluster model of water exerting hydrophobic interactions. The absorption and luminescence spectra and the fluorescence lifetimes have been determined.
Analytic expressions are derived for the eigenfunctions and eigenvalues of the Laplace transform and similar dilationally invariant integral equations of the first kind. Some generalised concepts of information theory are introduced to show how the use of these eigenfunctions enables the maximum possible amount of information to be obtained when solving the inverse problem numerically. These concepts also explain how the amount of information available depends on the level of noise in the calculation and on the structure of the particular integral kernel. Some numerical examples which illustrate these points are presented.
A description of the ab initio quantum chemistry package GAMESS is presented. Chemical systems containing atoms through radon can be treated with wave functions ranging from the simplest closed-shell case up to a general MCSCF case, permitting calculations at the necessary level of sophistication. Emphasis is given to novel features of the program. The parallelization strategy used in the RHF, ROHF, UHF, and GVB sections of the program is described, and detailed speecup results are given. Parallel calculations can be run on ordinary workstations as well as dedicated parallel machines. © John Wiley & Sons, Inc.
Comparative analysis of the absorption and fluorescence spectra and fluorescence excitation spectra of thioflavin T (ThT) in various solvents and in the composition of amyloid fibrils has shown that ThT, when excited in the region of the long-wavelength absorption band, fluoresces in the spectral region with a maximum at 478–484 nm. The appearance in aqueous and alcohol solutions of a fluorescence band with a maximum near 440 nm has been attributed to the presence in the composition of the ThT preparations of an impurity with an absorption band in the 340–350-nm range. The literature data showing that in glycerol ThT has a wide fluorescence spectrum with two maxima are due to the artifact connected with the use of a high concentration of the dye. It has been suggested that the cause of the low quantum yield of ThT aqueous and alcohol solutions is the breakage of the system of conjugated bonds due to the reorientation of the benzothiozole and benzaminic rings of ThT in the excited state with respect to one another. The main factor determining the high quantum yield of fluorescence of ThT incorporated in fibrils is the steric restriction of the rotation of the rings about one another under these conditions. The suggestions made have been verified by the quantum-chemical calculation of the ThT molecule geometry in the ground and excited states.
MOLEKEL is an interactive visualization ('postprocessing') program for molecular and electronic structure data, generating high-quality graphics for use in research and education. MOLEKEL has been ported to OpenGL and is now available on a number of platforms, including PC (http://www.cscs.ch/molekel/).
We describe details of its implementation, capabilities and the new added features.
Alzheimer’s disease (AD) and Parkinson’s disease are the most common forms of age-related neurodegenerative disorders. The pathogenesis of these and other neurodegenerative diseases remains unclear, and effective treatments are currently lacking. However, recent studies from three diverse disciplines, neuropathology, genetics, and biophysics, have begun to converge on a novel target for therapeutic attack: ordered protein aggregation. Indeed, abnormal protein aggregation characterizes many, if not all, neurodegenerative disorders, not just AD and Parkinson’s disease, but also Creutzfeldt–Jakob disease, motor neuron diseases, the large group of polyglutamine disorders, including Huntington’s disease (1), as well as diseases of peripheral tissue like familial amyloid polyneuropathy (FAP). Many of these deposits were originally identified by their histochemical staining property, hence their designation as amyloid (starch-like). Subsequently, it was learned that amyloid deposits contain extremely insoluble protein fibrils that share similar morphological features (80- to 150-Å fibrils) but comprise many different proteins with no obvious sequence similarity. This review will focus on biophysical studies of protein aggregation in AD and FAP, where mechanistic models connecting pathological and genetic data to clinical disease are beginning to emerge. These two examples illustrate two ends of the biophysical spectrum: in one (AD), a flexible peptide is poised to form fibrils, whereas in the other (FAP), a stable globular tetramer must dissociate and partially unfold before forming a new stable fibril structure.
Der für die geringe Fluoreszenzausbeute von Triphenylmethanfarbstoffen in flüssigen Lö sungen verantwortliche viskositätsabhängige strahlungslose Prozeϟ wird auf eine Rotation der Phenylgruppen zurückgeführt, die nicht frei, sondern unter der Wirkung eines Direktionsmoments erfolgt. Eine daraus abgeleitete Beziehung zwischen Fluoreszenzausbeute und Lö sungsmittelviskosität wird mit Messungen an Kristallviolett und anderen Verbindungen dieser Farbstoffklasse verglichen.
Photophysical parameters have been determined for coumarin laser dyes in a variety of organic solvents, water, and mixed media. The response of fluorescence emission yield and lifetime to changes in solvent polarity was a sensitive function of coumarin substitution pattern. Most important were substituent influences which resulted in enlarged excited-state dipole moments for the fluorescent state. For dyes displaying sharp reductions in emission yield and lifetime with increased solvent polarity, protic media and particularly water were most effective in inhibiting fluorescence. The temperature dependence of emission yield and lifetime was measured for two solvent-sensitive dyes in acetonitrile and in a highly viscous solvent, glycerol. The quenching of coumarin fluorescence by oxygen for dyes with lifetimes > 2 ns was also observed. The dominant photophysical features for coumarin dyes are discussed in terms of emission from an intramolecular charge-transfer (ICT) excited state and an important nonradiative decay path involving rotation of the amine functionality (7-position) leading to a twisted intramolecular CT state (TICT). The role of excited-state bond orders involving the rotating group in determining the importance of interconversions of the type ICT ..-->.. TICT is discussed. 73 references, 1 figure, 3 tables.
Pressure dependent measurements of the nonradiative torsional dynamics of triphenyl-methane dyes are reported. The viscosity dependence of the excited state absorption and fluorescence decays of crystal violet, dissolved in methanol, ethanol, and n-propanol, are studied over a viscosity range of between 0.5 and 500 cP (pressures up to 1.5 GPa ∼15 000 atm). The overall viscosity dependence of the excited state decay time is linear. The nonlinear viscosity dependencies observed in earlier studies may have resulted from the complex nature of the solvents used and, in the case of fluorescence quantum yield measurements, from a pressure dependence of the radiative rate. Differences between the viscosity dependence observed when varying pressure, temperature, or solvent type at low viscosities are tentatively ascribed to pressure and temperature induced conformational changes.
Despite the presence of a significant amount of carbohydrate in these fibrils, the staining reaction was eventually shown to be because of the protein component. The histologic benzothiazole dyes thioflavin S (ThS) and thioflavin T (ThT) under the appropriate conditions selectively stain amyloid structures in a number of pathological settings, as does the diazobenzidine sulfonate dye, Congo red, which is also birefringent when bound to fibrils. Phorwhite BBU, Sirius Red, and several other fluorescent and nonfluorescent aromatic molecules also show this property. Investigation of the amyloid fibril formation process requires not only the ability to distinguish the characteristic amyloid B-sheet structure from amorphous aggregates of the monomer or nonamyloid fibril forms of the precursor protein, but quantitation of the amyloid form as well. Congo red and thioflavin T undergo characteristic spectral alterations on binding to a variety of amyloid fibrils that do not occur on binding to the precursor polypeptides, monomers, or amorphous aggregates of peptide. Both dyes have been adapted to in vitro measurements of amyloid fibril formation.
A series of fluorescence probes (p-(N,N-dialkylamino)benzylidene malononitriles) which belong to a class of organic compounds known as 'molecular rotors' has been developed. The internal molecular rotation of these compounds can be slowed down by increasing the surrounding media rigidity, viscosity or decreasing the free-volume available for molecular relaxation. Inhibition of internal molecular rotation of the probe leads to a decrease in the non-radiative decay rate and consequently enhancement of fluorescence. Therefore, the fluorescence emission of this class of compounds allowed us to study both the static and dynamic changes in free-volume of polymers as a function of polymerization reaction parameters, molecular weight, stereoregularity, crosslinking, polymer chain relaxation and flexibility. In addition, the dependence of the fluorescence emission maximum of these probes on media polarity allow continuous monitoring of the probes location in the polymer matrix. Therefore, these fluorescence materials are capable of simultaneously probing the flexibility and polarity of the surrounding media.
An organic molecule isomerizes in viscous solvents when appropriate cavities are formed around it in the course of slow diffusive thermal fluctuations of solvent molecules. The isomerization occurs when fast twisting (vibrational) fluctuations around a bond get to have large amplitudes in such cavities. This situation can be described by the two-reaction-coordinate model of Sumi and Marcus originally proposed for electron transfer reactions. In fact, the rate constant derived from this model fits nicely to that observed for thermal Z→E isomerization of substituted azobenzenes and N-benzylideneanilines. The rate constant is influenced by slow speeds of diffusive motions of solvent molecules, whose relaxation time τ is usually proportional to the solvent viscosity η. It has a form of k = 1/(kTST−1+kf−1), where kTST, independent of τ, represents the rate constant expected from the transition state theory (TST), while kf ∝ τ−α with 0 < α ≤ 1 represents the part controlled by solvent fluctuations. An analytic expression of α for the isomerization reactions is given in terms of physical parameters underlying the reaction mechanism with cavity formation.
The interaction of thioflavin T, 3,6-dimethyl-2-(4-dimethylaminophenyl)-benzothiazolium cation (TFT) with DNA is studied using absorption and emission spectral methods. The observed hypochromism in the absorption spectra suggests that the TFT binds with DNA. The emission spectral changes show two different emission bands at 450 and 485nm for TFT in the presence of DNA. The emission intensity observed at 450nm decreases upon increasing the concentration of DNA. This further confirms the binding of TFT with DNA. The new emission band observed at 485nm for TFT increases with increasing the concentration of DNA. It is proposed that the binding of TFT with DNA facilitates the formation of emittive TFT dimer. An isoemissive point was observed at 464nm when the DNA concentration was higher than 1.35×10−5moldm−3. This observation indicates the conversion of monomer TFT to emittive TFT dimer. Both the absorption and emission spectral studies show that two types of bindings such as groove binding and electrostatic interaction are present in the TFT–DNA system. The addition of NaCl to TFT–DNA showed the removal of the electrostatic binding between TFT and DNA. The intensity of 485nm emission band is decreased upon the addition of NaCl to TFT–DNA system. The interaction of Na+ ions with the phosphate groups of the DNA blocked the electrostatic interaction of TFT cations with DNA. The emission band at 485nm shows only one type of interaction viz. the electrostatic interaction. The three-dimensional emission spectral studies shows only one contour due to monomer TFT emission in the absence of DNA. However, in the presence of DNA, TFT shows two contours due to monomer TFT and emittive TFT dimer.
The excimer emission of thioflavin T, 3,6-dimethyl-2-(4-dimethylaminophenyl)-benzothiazolium cation (TFT+), was observed in the presence of γ-CD at room temperature. The emission intensity initially decreased due to the formation of a 2:1 TFT+-γ-CD inclusion complex. At higher concentrations of γ-CD an increase in the emission intensity was observed due to the formation of a 2:2 TFT+-γ-CD inclusion complex. The formation of the 2:2 TFT+-γ-CD inclusion complex was responsible for the excimer emission. In the presence of β-CD, the TFT+ molecule did not show excimer emission. The excimer emission observed in the presence of γ-CD was analysed by excitation and 3-D spectral measurements.
Dual frequency pump–probe induced photoabsorption recovery is studied in two triphenylmethane dyes, malachite green and crystal violet, using femtosecond laser pulses. Comparison of the decays observed for different polarization conditions allows us to analyze the symmetry of electronic states. We observe that the simplest C2 or D3 symmetries cannot account for all our observations. In addition to the previously reported electronic relaxation decays, we observe two additional subpicosecond relaxation components. A first rising component is attributed to the conformational relaxation of torsional modes and a faster decay can be interpreted as originating from solvent limited vibrational relaxation. We attribute the solvent dependence of these relaxations to the nonrigid nature of the triphenylmethanes.
Time-resolved anisotropy measurements (TRAMS) have been used to study the aggregation of the β-amyloid (Aβ) peptide which is suspected of playing a central role in the pathogenesis of Alzheimer's Disease (AD). The experiments, which employ small quantities of fluorescently-labelled Aβ, in addition to the untagged peptide, have shown that the sensitive TRAMS technique detects the presence of preformed “seed” particles in freshly prepared solutions of Aβ. More importantly, as 100 μM solutions of Aβ containing tagged Aβ at a concentration level of either 0.5 or 1 μM are incubated, the TRAMS prove capable of detection of the peptide aggregation process through the appearance of a continuously increasing “residual anisotropy” within the time-resolved fluorescence data. The method detects Aβ aggregation in its earliest stages, well before complexation becomes apparent in more conventional methods such as the thioflavin T fluorescence assay. The TRAMS approach promises to provide a most attractive route for establishment of a high-throughput procedure for the early detection of the presence of amyloid aggregates in the screening of biological samples.
We present a theory which describes the effects of viscosity on those electronic relaxation processes in solution in which the intramolecular potential surface does not present a barrier to the motion leading to the decay of the initially formed excited state. We model the reactive motion as the motion of a solute particle on the excited state potential surface with a position dependent sink which gives rise to the decay of the excited state population. Three different types of sinks are considered: (A) a pinhole sink at the minimum of the potential surface; this models the situation when the molecule decays to ground state as soon as it reaches the potential minimum; (B) a Gaussian sink with probability of decay maximum at the potential minimum; (C) a Lorentzian sink with maximum decay at the potential minimum. For case (A) an explicit analytic solution is obtained for the decay rate, but for cases (B) and (C) we obtained the decay rate numerically. Model (A) predicts nonexponential decay at all viscosities except at long times when the decay is single exponential. For cases (B) and (C) the decay is single exponential at low viscosities but becomes multiexponential at high viscosities. We show that the experimentally observed fractional viscosity dependence of fluorescence quantum yield arises naturally in this theory due to the position dependence of the sink as well as due to the competition between radiative and nonradiative relaxation. Our model also predicts a crossover from an apparent negative (constant viscosity) activation energy at low viscosities to a positive activation energy at high viscosity. The physical significance of these results is discussed in light of the available experimental results on TPM dye relaxation. Some possible generalizations of our theory to more realistic cases are indicated.
Femtosecond fluorescence upconversion studies have been performed for auramine (a diphenylmethane dye), dissolved in ethanol, as a function of temperature. It is found that the (sub)picosecond decay components in the fluorescence slow down as the temperature is lowered from 293 K to 173 K. From the observation of a residual fluorescence, with a viscosity-dependent lifetime of about 30 ps (or longer at higher viscosity), and transient absorption results it is concluded that the two-state sink function model [B. Bagchi, G. R. Fleming, and D. W. Oxtoby, J. Chem. Phys. 78, 7375 (1983)] does not apply in the case of auramine. Comparison of the auramine fluorescence kinetics in ethanol and decanol shows that diffusional twisting and not solvation is the main cause for the (sub)picosecond excited state relaxation. To explain the experimental results, adiabatic coupling between a locally excited emissive state (F) and a nonemissive excited state (D) is considered. Torsional diffusion motions of the phenyl groups in the auramine molecule are held responsible for the population relaxation along the adiabatic potential of the mixed state, S1 (comprised of the F and D states). Simulation of the excited state dynamics is feasible assuming a barrierless-shaped potential energy for S1 and applying the Smoluchowski diffusion equation. The temporal behavior of the auramine band emission was simulated for the temperature range 293 K >T>173K, with the temperature, T, and the viscosity coefficient, η, being the only variable parameters. The simulated temporal behavior of the emission in the investigated temperature range is compatible with that obtained experimentally. The rotational diffusion coefficient for the auramine phenyl groups as extracted from the simulations is found to follow the Einstein-Stokes relation. From the numerical calculations the effective radius of the twisting phenyl groups is determined as 1.0 Å which compares well with the actual value of 1.2 Å.
The dependence of the fluorescence efficiency of the dyes crystal violet and auramine O on solvent viscosity at room temperature has been investigated using pressure to alter the viscosity. Pressures to 11 kbar on four polar solvents were utilized to obtain a 5 order of magnitude viscosity range. We confirm the η2/3 dependence of fluorescence intensity on viscosity shown for crystal violet by Förster and Hoffman [T.H. Förster and G. Hoffman, Z. Physik. Chem. NF, 75, 63(1971)] and observe the same dependence for auramine O over a smaller range of viscosities with a large deviation at low viscosity. The effect of increasing solvent viscosity on the peak shift and half‐width change is briefly discussed in terms of a configuration coordinate model.
The nonradiative decay dynamics of crystal violet, and other triphenyl‐methane dyes, dissolved in a variety of solvents, are studied as a function of temperature. A linear viscosity dependence of the excited state absorption decay time in n‐alcohol solvents is found at several constant temperatures. The temperature dependence at constant viscosity is anomalously negative over the entire viscosity range (0.6 to 8.0 cP) of these experiments. Various possible mechanisms for the observed behavior are critically discussed. Two color excite‐and‐probe studies reveal ground state bleach recovery times which are independent of excitation wavelength but strongly dependent on probe wavelength. The faster decay on the red side of the ground state absorption is shown to be the result of stimulated emission rather than the influence of a second state.
The theoretical predictions of three theories for large amplitude dynamics on a barrierless excited state potential are tested. The predicted forms for the time dependent integrated excited state population are compared with experimentally measured excited state absorption decays of crystal violet in various solvents. We have generalized the early theory of Oster–Nishijima [J. Am. Chem. Soc. 78, 1581 (1956)] to allow for arbitrary placement of the initial excited state population relative of the nonradiative sinks which couple the flat excited state potential to the ground state. An analytic form for decay of the integrated excited state population is derived for this generalized flat potential model. In addition we have found that the predictions of Forster–Hoffmann [Z. Phys. Chem. NF 75, 63 (1971)] cannot be made to fit the experimentally measured excited state decays of crystal violet. Finally we show that the theory of Bagchi–Fleming–Oxtoby [J. Chem. Phys. 78, 7375 (1983)] can fit the decays observed in various solvents fairly well. The best fits, however, are obtained using the generalized flat potential model.
Thioflavine T (ThT) interacts in tissue sections with amyloid deposits comprised of a variety of protein species giving a characteristic fluorescent complex. Binding of the dye to amyloid fibrils in suspension generates an amyloid-specific fluorescent signal. This interaction with amyloid fibrils formed from different polypeptides and proteins containing antiparallel β-pleated sheet secondary structure is selective, occurring strongly with amyloid fibrils formed from insulin, transthyretin, polyglycine(I), Aβ (1–40), and weakly with β2–microglobulin. No fluorescence changes were seen with β-sheet fibrillar poly-L-lysine, islet amyloid peptide (20–29), or poly-L-serine. Native forms of transthyretin, insulin, β2-microglobulin, poly-L-lysine, Aβ(1–40), or several proteins containing high percentages of β-sheet were also unreactive. The affinity of the amyloid fibrils for ThT varied: (apparent Kd's 0.033 – 10 μ Amyloid A protein <insulin <ApoAII <polyglycine <transthyretin <Aβ(1–40). The spectral changes induced by the different amyloid fibrils are qualitatively the same regardless of the pKa of the interaction with ThT or the identity of the amyloid fibrils, suggesting that the quaternary and secondary, but not the primary structure of the amyloid fibrils are important in forming the amyloid fibril-specific ThT species. Thus, Thioflavine T provides a useful tool for the investigation of amyloidogenesis in a variety of systems.
The dynamic behavior of a dye-in-polymer was revealed by electronic spectroscopy. Absorption, emission, and polarization spectra of [p-(dialkylamino)benzylidene]malononitrile 1 in poly(vinyl acetate) (PVAc) showed that the dye exists in monomeric form only at very low concentrations (<0.03 M). At higher concentrations and up to 0.6 M dye-in-polymer, dimeric species exists in equilibrium with the monomers, with an equilibrium association constant of 1.56 M-1. Further increase in dye concentration leads to the formation of larger aggregates. The fluorescence quantum yields, φf, of the dye monomer, dimer, and amorphous solid were ∼0.01, ∼0.1, and 0.066, respectively. The higher φf of the dimer was attributed to a decrease in the rate of radiationless decay of 1 due to restricted molecular relaxation resulting from the binding of two monomer molecules. The polarization ratios (P) of the monomer, dimer, and amorphous solid were 0.5, 0.2, and 0.03, respectively. The value of 0.5 for the monomer indicates parallel absorption and emission transition moments with no molecular motion in the polymer within the lifetime of the singlet excited state (∼30 ps).
A new intermediate neglect of differential overlap (INDO-SCF-CI) method capable of calculating configuration interaction for transition-metal complexes is described. The technique is characterized by the use of atomic spectroscopic information in the formation of one-center, one-electron matrix elements and in the evaluation of two-electron integrals. All one-centered integrals that mix upon geometric rotations are found to be essential for calculation of configuration interaction and are retained. The method is applied to the calculation of the photoelectric and electronic spectra of ferrocene. Triplet and singlet state d → d* and charge-transfer transitions are considered. Three nearly degenerate triplet states are calculated for ferrocene at ∼20 500 cm-1 as compared to an observed triplet state at 18 900 cm-1. Three singlet transitions of the d → d* type are calculated at 21 700, 23 900, and 31 900 cm-1, in very good accord with the experimental observations and assignments of 1E1″ at 21 800 cm-1, 1E2″ at 24 000 cm-1, and 1E1″ at 30 800 cm-1. The lowest charge-transfer excitation is calculated at 36 900 cm-1 compared to the experimental observation at 37 700 cm-1. A considerable number of states above 36 900 cm-1 are calculated and a possible assignment of the observed states in the higher energy region is given. The calculated energies allow spectral assignment in good agreement with experiment and help resolve prior ambiguities in the assignment. The relative energies calculated for the ionic states of the ferroceniun ion (3E2′ < 2A1′ < 2E1″) are in good accord with previous ab initio calculations. The first two of these states are formed from the formal loss of an electron from a metal orbital; the third, from the loss of an electron from a ligand orbital. For all of these states relaxation upon ionizatlon is so significant that the net charge on iron increases only negligibly from +1.9 in ferrocene to +2.0 in the ferrocene ion. This observation is also in agreement with ab initio findings and has some support from Mössbauer spectroscopy.
The spectroscopic and electrochemical properties of a series of p-N,N-dialkylaminobenzylidenemalononitriles derivatives (1, 2, and 3) have been studies. These materials exhibit a strong intramolecular charge-transfer absorption. In this series the conformational freedom of the electron-donating group (R2N) was progressively restricted by structurally anchoring the nitrogen atom to the aromatic ring by alkyl chains. Molecular restriction of rotation of the NR2 group had a profound effect on the nonradiative decay rate and the excited state dipole moment. However, it had little effect on the ground state dipole moment. The most pronounced change in dipole moment (Δμ) on electronic excitation was observed when the NR2 group was free to rotate. The rate of internal rotation of the dialkylamino group was estimated to be 2 × 1011 s-1.
The effect of solvent viscosity and temperature on the fluorescence quantum yield of p-(dimethylamino)-benzylidenemalononitrile (1) and julolidinemalononitrile (3) has been studied in ethyl acetate, dimethyl phthalate, and glycerol. In the low-viscosity solvent, ethyl acetate, when temperature and viscosity variations are studied, a hydrodynamic model with a stick boundary condition gives an accurate and consistent description of the dynamics of the torsional motion of the probes. However, in medium- and high-viscosity solvents deviations from the hydrodynamic model are seen. In these solvents the free-volume concept was found to provide an accurate description of the solvent viscosity-temperature behavior and the probe torsional relaxation dynamics.
Rate constants for chemical reactions limited by electron, proton, or atom-group transfer in a large molecular system are formulated for the case where the reaction coordinate is composed not only of intramolecular or atomic vibrational motions but also of conformational fluctuations of the system itself driven by thermal motions of solvent molecules. These fluctuations are diffusive, being describable in terms of Brownian motions. Their relaxation time τ regarded as proportional to the viscosity η of solvents is much longer than periods of the vibrational motions. In this situation it is shown that when reactant populations get to decay single exponentially after a time of order τ in exthothermic reactions, rate constant ks tends, irrespective of initial conditions, to the inverse of the first passage time for initially thermalized reactants. This ks can be expressed as 1/(ke-1 + kf-1), where ke represents the thermal equilibrium rate constant obtainable by the usual theories such as the transition-state theory, while kf represents a fluctuation-limited rate constant decreasing with increasing τ or η. When ke is written as ν exp(-ΔG*/kBT) with the height ΔG* of the transition state and the frequency factor ν at temperature T, we show kf ∼ τ-αν1-α exp(-γΔG*/kBT) with positive constants α and γ both smaller than unity. When ke ≪ kf in the small-τν limit, we get ks ≅ kc, covering the result of the usual theories. When kf ≪ ke in the large-τν limit, on the other hand, we get ks ≅ kf. This limit is consistent with recent observations that ks is proportional to η-α in some reactions, including biochemical ones. In this limit, ks (∝ ν1-α) depends only weakly on ν, which measures the strength of the matrix element causing the reaction. The reactant distribution is maintained in a nonthermal equilibrium form during the reaction.
IntroductionExperimentalResults and Discussion Auramine(a) Transient Absorption(b) Fluorescence Up-ConversionIonic Styryl DyesConclusion Auramine(a) Transient Absorption(b) Fluorescence Up-ConversionIonic Styryl Dyes
Steady-state and time-resolved fluorescences were studied in alcohol oxidase from methylotropic yeast in the presence of substrate (ethanol). The fluorescence decay was found to be non-exponential and to reflect the heterogeneity of the microenvironment of the emitting tryptophanyls.The fluorescence decay of FAD located in the active centre can be described by the sum of two exponential components. The reason for this is suggested to be the presence of two different coenzyme conformations related to alcohol oxidase. The intensity of FAD fluorescence considerably increased as the protein denaturated and could serve as a criterion of enzyme nativity.
Thioflavine T (ThT) associates rapidly with aggregated fibrils of the synthetic β/A4-derived peptides β(1–28) and β(1–40), giving rise to a new excitation (ex) (absorption) maximum at 450 nm and enhanced emission (em) at 482 nm, as opposed to the 385 nm (ex) and 445 nm (em) of the free dye. This change is dependent on the aggregated state as monomeric or dimeric peptides do not react, and guanidine dissociation of aggregates destroys the signal. There was no effect of high salt concentrations. Binding to the β(1–40) is of lower affinity, Kd 2 μM, while it saturates with a Kd of 0.54 μM for β(1–28). Insulin fibrils converted to a β-sheet conformation fluoresce intensely with ThT. A variety of polyhydroxy, polyanionic, or polycationic materials fail to interact or impede interaction with the amyloid peptides. This fluorometric technique should allow the kinetic elucidation of the amyloid fibril assembly process as well as the testing of agents that might modulate their assembly or disassembly.
We describe the theory and practical aspects of analyzing fluorescence anisotropy decays in terms of correlation times distributions. In our model the rotational motions of the fluorophores were described using Gaussian or Lorentzian distributions of the correlation times. The theory is presented both for time and frequency-domain measurements, although the simulations and measurements are focused on the frequency-domain measurements of the anisotropy decays. Analysis of simulated data is presented to illustrate the nature of the data and the resolution which can be expected with presently available frequency-domain measurements. Additionally, we describe experimental data for samples where one can reasonably expect a single exponential and/or discrete multi-exponential correlation time distributions, and for samples where the anisotropy decay might be expected to display a distribution of correlation times. These samples include small single tryptophan peptides in propylene glycol, the single tryptophan residue in S. Nuclease, and the single tryptophan residue in the native and partially unfolded states of ribonuclease T1.
Fluorescence decay curves exhibiting good deconvolution fits to two or three exponentials can be interpreted in terms of two or three distinct lifetimes or as the result of complex underlying distributions which are not evident because of statistical noise. This work deals with the recovery of complex distributions of lifetimes from single photon decay data through the use of an exponential series probe function with fixed lifetimes and variable pre-exponential coefficients.
Most algorithms for the least-squares estimation of non-linear parameters have centered about either of two approaches. On the one hand, the model may be expanded as a Taylor series and corrections to the several parameters calculated at each iteration on the assumption of local linearity. On the other hand, various modifications of the method of steepest-descent have been used. Both methods not infrequently run aground, the Taylor series method because of divergence of the successive iterates, the steepest-descent (or gradient) methods because of agonizingly slow convergence after the first few iterations. In this paper a maximum neighborhood method is developed which, in effect, performs an optimum interpolation between the Taylor series method and the gradient method, the interpolation being based upon the maximum neighborhood in which the truncated Taylor series gives an adequate representation of the nonlinear model. The results are extended to the problem of solving a set of nonlinear algebraic e
We used a fluorometric method to examine amyloid fibrils, in vitro. These fibrils in the case of both murine senile and secondary amyloidosis were purified to apparent homogeneity from the water-suspended fraction of the liver of senescence-accelerated mouse, using sucrose density ultracentrifugation, and then the following assays were performed. In the absence of amyloid fibrils, thioflavine T fluoresced faintly at the excitation and emission maxima of 350 and 438 nm, respectively. In the presence of amyloid fibrils, thioflavine T fluoresced brightly at the excitation and emission maxima of 450 and 482 nm, respectively, and the fluorescence change was linear from 0 to 2.0 micrograms/ml amyloid fibrils. This fluorescence was maximal around pH 9.0. Fluorescence intensity in the presence of a constant amount of amyloid fibrils reached a plateau with increase in the thioflavine T concentration. Normal high density lipoproteins which contain apo A-II, the precursor of amyloid fibrils in murine senile amyloidosis, and acute phase high density lipoproteins which contain serum amyloid protein A, the precursor of amyloid fibrils in secondary amyloidosis, showed little fluorescence. The fluorescence was considerably diminished when structure of the amyloid fibrils was disrupted by guanidine-HCl treatment. This method will be useful for the determination of amyloid fibrils in vitro.
9-(Dicyanovinyl)julolidine (DCVJ) is a fluorescent dye whose intramolecular rotational relaxation is solvent dependent. Since its quantum yield increases with decreasing free volume, this molecule has been very useful in monitoring synthetic polymer reactions and measuring local microviscosity changes in phospholipid bilayers [Loutfy, R. O. (1986) Pure Appl. Chem. 58, 1239-1248; Kung, C. E., & Reed, J. K. (1986) Biochemistry 25, 6114-6121]. We have used DCVJ to follow the polymerization of tubulin, a protein that can assemble into a variety of polymorphic microstructures. DCVJ binding to free tubulin is accompanied by an increase in quantum yield, indicating that DCVJ has become partially immobilized. At 4 degrees C, DCVJ binds to a single population of high-affinity hydrophobic sites (Kd = 1.12 +/- 0.26 microM) with a stoichiometry that is protein concentration dependent. n, the number of moles of DCVJ bound per mole of alpha beta dimer, approaches 1 at concentrations less than or equal to 0.5 mg/mL but decreases to a lower limit of approximately 0.3 at concentrations greater than or equal to 2.0 mg/mL. The quantum yield also increases with increasing protein concentration. This trend is unaltered by the presence of microtubule-associated proteins. These results are analyzed in terms of a concentration-dependent oligomerization of tubulin at 4 degrees C. When tubulin is polymerized at 37 degrees C to microtubules or to sheets in the presence of Zn2+, the fluorescence intensity of DCVJ increases although the magnitude of this increase differs significantly. We are able to use the distinct fluorescent and binding characteristics of the bound dye to distinguish between these two polymorphs on a molecular level.
The decay of the tryptophanyl emission in proteins is often complex due to the sensitivity of the tryptophan excited state to its surroundings. The traditional analysis of the decay curve using exponential components is based on the identification of each component with a particular protein conformation. An alternative approach assumes that proteins can exhibit a large number of conformations and that, at room temperature, the interconversion rate between conformations can be of the same order of magnitude as the excited-state decay rate. Following this assumption, the analysis of the protein emission was performed using continuous distributions of lifetime values. The number of average protein conformations, the range of mobility around each conformation, and the rate of interconversion between conformations determines the characteristics of the lifetime distribution. The fluorescence decay from some single tryptophan proteins was measured using multifrequency phase fluorometry and analyzed using a sum of exponentials, unimodal and bimodal probability-density functions, and the analytical form for lifetime distribution obtained for a model in which the tryptophan residue can move in a single potential well. For ribonuclease T1 and neurotoxin variant 3, the sum of two exponentials and bimodal probability-density functions gave comparable results, whereas for phospholipase A2, the description of the decay required three exponentials or bimodal probability-density functions. Also the temperature dependence of the fluorescence decay was investigated. It was found that the lifetime distribution was broader and shifted toward longer lifetime values at lower temperature. The analysis of the decay of tryptophan in buffer and of some tryptophan derivatives gave single-exponential decays. The single-potential well lifetime distribution, which has only three adjustable parameters, gave good fits for all cases investigated, but in the case of phopholipase A2, the temperature dependence of the parameters that describe the single-potential well distribution indicated the inadequacy of this model at lower temperature, suggesting that multiple potential wells can describe better the decay for this protein.
Analysis of fluorescence decay kinetics aims at the determination of the analytic expression and the numerical values of the pertinent parameters which describe the decay process. In the well-known method of least-squares, one assumes a plausible functional form for the decay data and adjusts the values of the parameters until the statistically best fit is obtained between the data and the calculated decay function, i.e., until the sum of the weighted squares of the residuals is at a minimum. It is shown that proper weighting of the squares of the residuals may markedly improve the quality of the analysis. Such weighting requires information about the character of the experimental noise, which is often available, e.g., when the noise is due to counting error in photon-counting techniques. Furthermore, dramatic improvements in the accuracy of the analysis may often be achieved by use of auxiliary information available about the system studied. For example, the preexponents in a multiexponential fluorescence decay of a mixture of chromophores (such as tryptophan residues in a protein molecule) may sometimes be estimated independently; much higher accuracy can then be attained for the decay lifetimes by analysis of the decay kinetics. It is proposed that the shape of the autocorrelation function of the weighted residuals may serve as a convenient criterion for the quality of fit between the experimental data and the decay function obtained by analysis. The above conclusions were reached by analysis of computer-simulated experiments, and the usefulness of this approach is illustrated. The importance of stating the uncertainties in the estimated parameters inherent in the analysis of decay kinetics is stressed.
The benzothiazole dye thioflavin T (ThT) is a classical amyloid stain for senile plaques containing beta/A4 peptide in Alzheimer's disease brain. ThT also binds rapidly and specifically to the anti-parallel beta-sheet fibrils formed from synthetic beta (1-40) peptide, but does not bind to monomer or oligomeric intermediates. The fibrillar beta-sheet-bound dye species undergoes a characteristic 120 nm red shift of its excitation spectrum that may be selectively excited at 450 nm, resulting in a fluorescence signal at 482 nm. Mixing of preformed beta (1-40) amyloid fibrils with ThT in a stopped-flow spectrophotometer, monitoring fluorescence emission at > 475 nm while exciting at 450 nm, distinguished multiple kinetic phases of roughly equivalent amplitude with tau's in the ranges of 0.007, 0.05, 0.75, and 10-20 s. The fastest reaction appears to reflect a bimolecular dye binding event while the remaining reactions are rate-limited by protein tertiary or quaternary conformational changes. The high activation energies of the three slower reactions support this interpretation. The ThT concentration dependence of the reaction rates at different ratios of ThT/beta (1-40) amyloid fibrils rules out a rate-limiting conformational change occurring prior to ligand binding. ThT is a useful probe for the aggregated fibrillar state of beta (1-40) amyloid fibrils as the amyloid-specific fluorescence reports only fibrillar species. The binding of ThT does not interfere with the aggregation of this peptide into amyloid fibrils. The putative conformational changes detected by the ThT fluorescence suggest that small pharmacologic ligands can perturb and possibly dissociate A beta amyloid fibrils.
The assembly and misassembly of normally soluble proteins into fibrilar structures is thought to be a causative agent in a variety of human amyloid and prion diseases. Structural and mechanistic studies of this process are beginning to elucidate the conformational changes required for the conversion of a normally soluble and functional protein into a defined quaternary structure.
Amyloid plaques composed of the fibrillar form of the amyloid-beta protein (Abeta) are the defining neuropathological feature of Alzheimer's disease (AD). A detailed understanding of the time course of amyloid formation could define steps in disease progression and provide targets for therapeutic intervention. Amyloid fibrils, indistinguishable from those derived from an AD brain, can be produced in vitro using a seeded polymerization mechanism. In its simplest form, this mechanism involves a cooperative transition from monomeric Abeta to the amyloid fibril without the buildup of intermediates. Recently, however, a transient species, the Abeta amyloid protofibril, has been identified. Here, we report studies of Abeta amyloid protofibril and its seeded transition into amyloid fibrils using atomic force microscopy.
Seeding of the protofibril-to-fibril transition was observed. Preformed fibrils, but not protofibrils, effectively seeded this transition. The assembly state of Abeta influenced the rate of seeded growth, indicating that protofibrils are fibril assembly precursors. The handedness of the helical surface morphology of fibrils depended on the chirality of Abeta. Finally, branched and partially wound fibrils were observed.
The temporal evolution of morphologies suggests that the protofibril-to-fibril transition is nucleation-dependent and that protofibril winding is involved in that transition. Fibril unwinding and branching may be essential for the post-nucleation growth process. The protofibrillar assembly intermediate is a potential target for AD therapeutics aimed at inhibiting amyloid formation and AD diagnostics aimed at detecting presymptomatic disease.