Photophysical Properties of Fluorescent Probe Thioflavin T in Crowded Milieu

Thioflavin T (ThT) is a widely used fluorescent probe of amyloid fibrils, which accompanies many serious neurodegenerative and other diseases. Until recently, examinations of processes of amyloid fibril formation in vitro were conducted in solutions whose properties were significantly different from those found inside the densely packed cells. Such crowded cellular milieu is typically simulated in vitro using concentrated solutions of inert polymers, which do not usually interact with proteins. However, these crowding agents can have a direct effect on the ThT molecule, and this effect must be taken into account. We examined the influence of PEG-400, PEG-12000, and Dextran-70 on the photophysical properties of ThT. It was shown that these crowding agents caused the red shift of the absorption, fluorescence excitation, and fluorescence spectra of ThT. Under these conditions, the increases of the molar extinction coefficient, fluorescence quantum yield, and excitation lifetime of ThT are also observed. However, these changes are significantly less pronounced than those observed for ThT bound to fibrils. It is concluded that, despite some effects of crowding agents on intrinsic fluorescent properties of ThT, this dye can be used as a probe of structure and formation of amyloid fibrils in crowded milieu in vitro

Analyzing Thioflavin T Binding to Amyloid Fibrils by an Equilibrium Microdialysis-Based Technique

A new approach for the determination of the amyloid fibril – thioflavin T (ThT) binding parameters (the number of binding modes, stoichiometry, and binding constants of each mode) is proposed. This approach is based on the absorption spectroscopy determination of the concentration of free and bound to fibril dye in solutions, which are prepared by equilibrium microdialysis. Furthermore, the proposed approach allowed us, for the first time, to determine the absorption spectrum, molar extinction coefficient, and fluorescence quantum yield of the ThT bound to fibril by each binding modes. This approach is universal and can be used for determining the binding parameters of any dye interaction with a receptor, such as ANS binding to proteins in the molten globule state or to protein amorphous aggregates

Point mutations affecting yeast prion propagation change the structure of its amyloid fibrils

We investigated the effect of the point substitutions in the N-terminal domain of the yeast prion protein Sup35 (Sup35NMp) on the structure of its amyloid fibrils. As the objects of the study, proteins with mutations that have different influence on the [PSI+] prion propagation, but do not prevent the aggregation of Sup35NMp in vitro were chosen. The use of the wide range of physico-chemical methods allowed us to show significant differences in the structure of these aggregates, their physical size, clumping tendency. Also we demonstrated that the fluorescent probe thioflavin T (ThT) can be successfully used for investigation of subtle changes in the structural organization of fibrils formed from various Sup35NMp. The obtained results and our theoretical predictions allowed us to conclude that some of selected amino acid substitutions delimit the region of the protein that forms the core of amyloid fibrils, and change the fibrils structure. The relationship of structural features of in vitro Sup35NMp amyloid aggregates with the stability of the [PSI+] prion in vivo allowed us to suggest that oligopeptide repeats (R) of the amyloidogenic N-terminal domain of Sup35NMp from R0 to R2 play a key role in protein aggregation. Their arrangement rather than just presence is critical for propagation of the strong [PSI+] prion variants. The results confirm the suitability of the proposed combination of theoretical and empirical approaches for identifying changes in the amyloid fibrils structure, which, in turn, can significantly affect both the functional stability of amyloid fibrils and their pathogenicity.Laboratorio de Investigación y Desarrollo de Bioactivo

Fluorescence Quantum Yield of Thioflavin T in Rigid Isotropic Solution and Incorporated into the Amyloid Fibrils

In this work, the fluorescence of thioflavin T (ThT) was studied in a wide range of viscosity and temperature. It was shown that ThT fluorescence quantum yield varies from 0.0001 in water at room temperature to 0.28 in rigid isotropic solution (T/η→0). The deviation of the fluorescence quantum yield from unity in rigid isotropic solution suggests that fluorescence quantum yield depends not only on the ultra-fast oscillation of ThT fragments relative to each other in an excited state as was suggested earlier, but also depends on the molecular configuration in the ground state. This means that the fluorescence quantum yield of the dye incorporated into amyloid fibrils must depend on its conformation, which, in turn, depends on the ThT environment. Therefore, the fluorescence quantum yield of ThT incorporated into amyloid fibrils can differ from that in the rigid isotropic solution. In particular, the fluorescence quantum yield of ThT incorporated into insulin fibrils was determined to be 0.43. Consequently, the ThT fluorescence quantum yield could be used to characterize the peculiarities of the fibrillar structure, which opens some new possibilities in the ThT use for structural characterization of the amyloid fibrils

β-Barrels and Amyloids: Structural Transitions, Biological Functions, and Pathogenesis

Insoluble protein aggregates with fibrillar morphology called amyloids and β-barrel proteins both share a β-sheet-rich structure. Correctly folded β-barrel proteins can not only function in monomeric (dimeric) form, but also tend to interact with one another—followed, in several cases, by formation of higher order oligomers or even aggregates. In recent years, findings proving that β-barrel proteins can adopt cross-β amyloid folds have emerged. Different β-barrel proteins were shown to form amyloid fibrils in vitro. The formation of functional amyloids in vivo by β-barrel proteins for which the amyloid state is native was also discovered. In particular, several prokaryotic and eukaryotic proteins with β-barrel domains were demonstrated to form amyloids in vivo, where they participate in interspecies interactions and nutrient storage, respectively. According to recent observations, despite the variety of primary structures of amyloid-forming proteins, most of them can adopt a conformational state with the β-barrel topology. This state can be intermediate on the pathway of fibrillogenesis (“on-pathway state”), or can be formed as a result of an alternative assembly of partially unfolded monomers (“off-pathway state”). The β-barrel oligomers formed by amyloid proteins possess toxicity, and are likely to be involved in the development of amyloidoses, thus representing promising targets for potential therapy of these incurable diseases. Considering rapidly growing discoveries of the amyloid-forming β-barrels, we may suggest that their real number and diversity of functions are significantly higher than identified to date, and represent only “the tip of the iceberg”. Here, we summarize the data on the amyloid-forming β-barrel proteins, their physicochemical properties, and their biological functions, and discuss probable means and consequences of the amyloidogenesis of these proteins, along with structural relationships between these two widespread types of β-folds

Reevaluation of ANS binding to human and bovine serum albumins: key role of equilibrium microdialysis in ligand - receptor binding characterization.

In this work we return to the problem of the determination of ligand-receptor binding stoichiometry and binding constants. In many cases the ligand is a fluorescent dye which has low fluorescence quantum yield in free state but forms highly fluorescent complex with target receptor. That is why many researchers use dye fluorescence for determination of its binding parameters with receptor, but they leave out of account that fluorescence intensity is proportional to the part of the light absorbed by the solution rather than to the concentration of bound dye. We showed how ligand-receptor binding parameters can be determined by spectrophotometry of the solutions prepared by equilibrium microdialysis. We determined the binding parameters of ANS - human serum albumin (HSA) and ANS - bovine serum albumin (BSA) interaction, absorption spectra, concentration and molar extinction coefficient, as well as fluorescence quantum yield of the bound dye. It was found that HSA and BSA have two binding modes with significantly different affinity to ANS. Correct determination of the binding parameters of ligand-receptor interaction is important for fundamental investigations and practical aspects of molecule medicine and pharmaceutics. The data obtained for albumins are important in connection with their role as drugs transporters

A New Trend in The Experimental Methodology for The Analysis of The Thioflavin T Binding to Amyloid Fibrils

The studies on the determination of the characteristics of the amyloid fibril interaction with the dye were based on the analysis of the dependence of the ThT fluorescence intensity on its concentration in the solution containing the amyloid fibrils. In the present work, we revealed that this intuitive approach provided erroneous data. We propose a new approach which provides a means for characterizing the interaction of thioflavin T (ThT) with amyloid fibrils and for determining the binding stoichiometry and binding constants, absorption spectrum, molar extinction coefficient, and fluorescence quantum yield of the ThT bound to the sites of different binding modes of fibrils. The key point of this approach is sample preparation by equilibrium microdialysis. The efficiency of the proposed approach is demonstrated via the examination of the ThT binding to insulin and Aβ42 fibrils as well as to the native form of the Electrophorus electricus acetylcholinesterase. We show that the peculiarities of ThT interaction with amyloid fibrils depend on the amyloidogenic protein and on the binding mode. This approach is universal and can be used for the analysis of binding mechanism of any dye that interacts with its receptor. Therefore, the proposed approach represents an important addition to the existing arsenal of means for the diagnostics and therapy of the neurodegenerative diseases

Binding Stoichiometry and Affinity of Fluorescent Dyes to Proteins in Different Structural States

Protocol of determination of binding stoichiometry and affinity of fluorescent dyes with proteins in different structural states is proposed. The proposed approach is based on the spectrophotometric determination of concentrations of dye bound to protein and free dye in solutions prepared by equilibrium microdialysis. This technique allows also determining spectral properties of the bound dyes. The restrictions of the use of dye fluorescence intensity for characterization of its interaction with the target protein are discussed. It is shown that the dependence of the dye fluorescence intensity on its optical density together with the data on its binding parameter can give information about the dye fluorescence quantum yield. All procedures are illustrated by interaction of 8-anilino-1-naphthalenesulfonate (ANS) with bovine serum albumin

The dependences of fluorescence intensity of quinine sulfate (QS) on its optical density.

<p> are calculated values and are experimentally recorded and normalized values of fluorescence intensity of QS. Dots are the limiting value of at . The dependences of calculated values and experimentally determined values on total optical density are given in the Insert. Strait line is the dependence of on ().</p