Possible Mechanism Of Amyloid Formation By Apomyoglobin Mutants

Abstract

It is known that even proteins, not involved into diseases, are able to form amyloid-like structures similar in final architecture of their fibrils. This fact suggests that formation of aggregated cross-beta structure is a common property of a polypeptide chain under appropriate conditions. Sperm whale apomyoglobin was used to investigate amyloid formation because the properties and folding process of this protein are well known. Process of the apomyoglobin mutant aggregation was monitored under conditions close to physiological ones (40°C, pH 5.5) by ThT binding, turbidity, FTIR spectroscopy and electron microscopy. Mutated proteins contained a single point substitution at positions Val10 and Met131 by Ala, Phe and Trp. It was shown that the WT apomyoglobin formed aggregates not containing beta-structure, while variants of apomyoglobin have shown significant increase of ThT fluorescence intensity and changes in a form of FTIR spectra. These changes evidenced appearance of beta-structured aggregates, and EM images showed fibril-like aggregates. Kinetics of amyloid formation monitored by turbidity and ThT binding allowed to calculate three rate constants of amyloid formation and to distinguish three stages of this process. Obtained results suggest that the rate of the first stage is affected by a position of substitution, and is not influenced by its type. In contrast, the rate of the second stage depends on a type of substitution: it is slower for mutants with aromatic amino acid substitutions. This work was supported by INTAS grant 05-1000004-7747, partly by the Howard Hughes Medical Institute Award 55005607 to A.V. Finkelstein and by the RAS Program on “Molecular and Cellular Biology”.