Aggregation of Partially Unfolded Myosin Subfragment-1 into Spherical Oligomers with Amyloid-Like Dye-Binding Properties

• Published in: Journal of biochemistry (Tokyo) Vol. 139; no. 6; pp. 989 - 996
• Main Authors: Komatsu, Hideyuki, Shinotani, Nami, Kimori, Yoshitaka, Tokuoka, Jun-ichiro, Kaseda, Kuniyoshi, Nakagawa, Hiroyuki, Kodama, Takao
• Format: Journal Article
• Language: English
• Published: England Oxford University Press 01.06.2006
• Subjects: 3-morpholinopropanesulfonic acid; 4-bis (2-ethanesulfonic acid); amyloid; Amyloid - chemistry; Amyloid - metabolism; Animals; Chromatography, Gel - methods; circular dichroism; Circular Dichroism - methods; Congo Red - chemistry; dithiothreitol; DTT; high performance liquid chromatography; Hot Temperature; HPLC; Kinetics; Microscopy, Electron, Scanning - methods; MOPS; myosin subfragment 1; Myosin Subfragments - chemistry; Myosin Subfragments - metabolism; Myosin Subfragments - ultrastructure; Oligopeptides - chemistry; Oligopeptides - metabolism; partial unfolding; Particle Size; Peptide Fragments - chemistry; Peptide Fragments - metabolism; piperazine-1; PIPES; protein aggregation; Protein Conformation; Protein Denaturation; Protein Folding; Protein Structure, Secondary; Rabbits; SDS-PAGE; sodium dodecyl sulfate–polyacrylamide gel electrophoresis; Thermodynamics; Thioflavin T; ThioT; Time Factors
• ISSN: 0021-924X; 1756-2651
• DOI: 10.1093/jb/mvj111

Proteolytic myosin subfragment 1 (S1) is known to be partially unfolded in its 50-kDa subdomain by mild heat treatment at 35°C [Burke et al. (1987) Biochemistry 26, 1492-1496]. Here, we report that this partial unfolding is accompanied by aggregation of S1 protein. Characteristics of the aggregate thus formed were: (i) formation of transparent sediment under centrifugation at 183,000 x g; (ii) amyloid-like, dye-binding properties such as Congo red-binding and Thioflavin T fluorescence enhancement; (iii) a uniformly sized spherical appearance in electron micrographs; and (iv) sensitivity to tryptic digestion. Gel filtration analysis of the aggregation process indicates that the spheroid was formed through an intermediate oligomeric stage. The aggregate inhibited spontaneous aggregation of an isolated 50 kDa fragment into a large amorphous mass. The remaining native regions in the partially unfolded S1 were probably responsible for this effect. These results show that, unlike the 50-kDa fragment, the partially unfolded S1 molecules do not form amorphous aggregates but assemble into spherical particles. The native regions in partially unfolded S1 may be a determinant of aggregate morphology.