Dataset Information

Structural Features of Amyloid Fibrils Formed from the Full-Length and Truncated Forms of Beta-2-Microglobulin Probed by Fluorescent Dye Thioflavin T.

ABSTRACT: The persistence of high concentrations of beta-2-microglobulin (?2M) 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 ?2M in the tissues and biological fluids of patients. In this paper, the amyloid fibrils formed from the full-length ?2M (?2m) and its variants that lack the 6 and 10 N-terminal amino acids of the protein polypeptide chain (?N6?2m and ?N10?2m, 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 ?2M amyloid fibrils with affinity ~10? 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 ?2M 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-?2M fibrils binding parameters and characteristics of the bound dye allowed to prove not only the difference of the ?N10?2m fibrils from other ?2M fibrils (that can be detected visually, for example, by transmission electron microscopy (TEM), but also the differences between ?2m and ?N6?2m 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 ?2M amyloid fibrils. Information about amyloidogenic protein sequences can be claimed in the development of ways to inhibit ?2M fibrillogenesis for the treatment of dialysis-related amyloidosis.

SUBMITTER: Sulatskaya AI

PROVIDER: S-EPMC6164334 | biostudies-literature | 2018 Sep

REPOSITORIES: biostudies-literature

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Structural Features of Amyloid Fibrils Formed from the Full-Length and Truncated Forms of Beta-2-Microglobulin Probed by Fluorescent Dye Thioflavin T.

Sulatskaya Anna I AI Rodina Natalia P NP Polyakov Dmitry S DS Sulatsky Maksim I MI Artamonova Tatyana O TO Khodorkovskii Mikhail A MA Shavlovsky Mikhail M MM Kuznetsova Irina M IM Turoverov Konstantin K KK

International journal of molecular sciences 20180914 9

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 (ΔN ...[more]

PMID: 30223436

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Project description:The ?E693 (Japanese) mutation of the ?-amyloid precursor protein leads to production of ?E22-A? peptides such as ?E22-A?(1-39). Despite reports that these peptides do not form fibrils, here we show that, on the contrary, the peptide forms fibrils essentially instantaneously. The fibrils are typical amyloid fibrils in all respects except that they cause only low levels of thioflavin T (ThT) fluorescence, which, however, develops with no lag phase. The fibrils bind ThT, but with a lower affinity and a smaller number of binding sites than wild-type (WT) A?(1-40). Fluorescence depolarization confirms extremely rapid aggregation of ?E22-A?(1-39). Size exclusion chromatography (SEC) indicates very low concentrations of soluble monomer and oligomer, but only in the presence of some organic solvent, e.g., 2% (v/v) DMSO. The critical concentration is approximately 1 order of magnitude lower for ?E22-A?(1-39) than for WT A?(1-40). Several lines of evidence point to an altered structure for ?E22-A?(1-39) compared to that of WT A?(1-40) fibrils. In addition to differences in ThT binding and fluorescence, PITHIRDS-CT solid-state nuclear magnetic resonance (NMR) measurements of ?E22-A?(1-39) are not compatible with the parallel in-register ?-sheet generally observed for WT A?(1-40) fibrils. X-ray fibril diffraction showed different D spacings: 4.7 and 10.4 Å for WT A?(1-40) and 4.7 and 9.6 Å for ?E22-A?(1-39). Equimolar mixtures of ?E22-A?(1-39) and WT A?(1-40) also produced fibrils extremely rapidly, and by the criteria of ThT fluorescence and electron microscopic appearance, they were the same as fibrils made from pure ?E22-A?(1-39). X-ray diffraction of fibrils formed from 1:1 molar mixtures of ?E22-A?(1-39) and WT A?(1-40) showed the same D spacings as fibrils of the pure mutant peptide, not the wild-type peptide. These findings are consistent with extremely rapid nucleation by ?E22-A?(1-39), followed by fibril extension by WT A?(1-40), and "conversion" of the wild-type peptide to a structure similar to that of the mutant peptide, in a manner reminiscent of the prion conversion phenomenon.

Dataset Information

Structural Features of Amyloid Fibrils Formed from the Full-Length and Truncated Forms of Beta-2-Microglobulin Probed by Fluorescent Dye Thioflavin T.

Publications

Structural Features of Amyloid Fibrils Formed from the Full-Length and Truncated Forms of Beta-2-Microglobulin Probed by Fluorescent Dye Thioflavin T.

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