No abstract

In this work, α-synuclein amyloid fibrils—the formation of which is a biomarker of Parkinson’s disease—were investigated using the fluorescent probe thioflavin T (ThT). The experimental conditions of protein fibrillogenesis were chosen so that a sufficient number of continuous measurements could be performed to characterize and analyze all stages of this process. The reproducibility of fibrillogenesis and the structure of the obtained aggregates (which is a critical point for further investigation) were proven using a wide range of physical-chemical methods. For the determination of ThT-α-synuclein amyloid fibril binding parameters, the sample and reference solutions were prepared using equilibrium microdialysis. By utilizing absorption spectroscopy of these solutions, the ThT-fibrils binding mode with a binding constant of about 104 M−1 and stoichiometry of ThT per protein molecule of about 1:8 was observed. Fluorescence spectroscopy of the same solutions with the subsequent correction of the recorded fluorescence intensity on the primary inner filter effect allowed us to determine another mode of ThT binding to fibrils, with a binding constant of about 106 M−1 and stoichiometry of about 1:2500. Analysis of the photophysical characteristics of the dye molecules bound to the sites of different binding modes allowed us to assume the possible localization of these sites. The obtained differences in the ThT binding parameters to the amyloid fibrils formed from α-synuclein and other amyloidogenic proteins, as well as in the photophysical characteristics of the bound dye, confirmed the hypothesis of amyloid fibril polymorphism.

The persistence of high concentrations of beta-2-microglobulin (β2M) in the blood of patients with acute renal failure leads to the development of the dialysis-related amyloidosis. This disease manifests in the deposition of amyloid fibrils formed from the various forms of β2M in the tissues and biological fluids of patients. In this paper, the amyloid fibrils formed from the full-length β2M (β2m) and its variants that lack the 6 and 10 N-terminal amino acids of the protein polypeptide chain (ΔN6β2m and ΔN10β2m, respectively) were probed by using the fluorescent dye thioflavin T (ThT). For this aim, the tested solutions were prepared via the equilibrium microdialysis approach. Spectroscopic analysis of the obtained samples allowed us to detect one binding mode (type) of ThT interaction with all the studied variants of β2M amyloid fibrils with affinity ~104 M−1. This interaction can be explained by the dye molecules incorporation into the grooves that were formed by the amino acids side chains of amyloid protofibrils along the long axis of the fibrils. The decrease in the affinity and stoichiometry of the dye interaction with β2M fibrils, as well as in the fluorescence quantum yield and lifetime of the bound dye upon the shortening of the protein amino acid sequence were shown. The observed differences in the ThT-β2M fibrils binding parameters and characteristics of the bound dye allowed to prove not only the difference of the ΔN10β2m fibrils from other β2M fibrils (that can be detected visually, for example, by transmission electron microscopy (TEM), but also the differences between β2m and ΔN6β2m fibrils (that can not be unequivocally confirmed by other approaches). These results prove an essential role of N-terminal amino acids of the protein in the formation of the β2M amyloid fibrils. Information about amyloidogenic protein sequences can be claimed in the development of ways to inhibit β2M fibrillogenesis for the treatment of dialysis-related amyloidosis.

Hsp26 is a small heat shock protein (sHsp) from S. cerevisiae. Its chaperone activity is activated by oligomer dissociation at heat shock temperatures. Hsp26 contains 9 phosphorylation sites in different structural elements. Our analysis of phospho-mimetic mutations shows that phosphorylation activates Hsp26 at permissive temperatures. The cryo-EM structure of the Hsp26 40mer revealed contacts between the conserved core domain of Hsp26 and the so-called thermosensor domain in the N-terminal part of the protein, which are targeted by phosphorylation. Furthermore, several phosphorylation sites in the C-terminal extension, which link subunits within the oligomer, are sensitive to the introduction of negative charges. In all cases, the intrinsic inhibition of chaperone activity is relieved and the N-terminal domain becomes accessible for substrate protein binding. The weakening of domain interactions within and between subunits by phosphorylation to activate the chaperone activity in response to proteotoxic stresses independent of heat stress could be a general regulation principle of sHsps.