Search

Cytotoxicity of amyloid fibrils has been shown to depend on their structure. However, specific features of toxic and non-toxic amyloids remain unclear. Here we focus on the relationship between structural characteristics of the fibrils and their cytotoxicity. Bovine carbonic anhydrase B (BCAB) serves as the object of this study because its amyloids reduce cell viability. Limited proteolysis and mass spectrometry were used to determine BCAB regions forming the core of amyloid fibrils. Four BCAB mutants with substitutions reducing hydrophobicity in the regions important for amyloid formation were obtained to study the kinetics of aggregation, structural features, and cytotoxicity of the amyloids. We demonstrate that fibrils of WT BCAB, L78A, L139A, and M239A variants display a pronounced toxic effect on eukaryotic cells, while I208A mutation significantly reduces the cell-damaging effect of amyloids. The data obtained conclude that cytotoxicity of BCAB fibrils does not depend on their length, secondary structure, and exposure of hydrophobic groups to the solvent. A distinctive feature of the low-toxic I208A fibrils is their specific morphology characterized by the lateral protofilaments association and formation of fibril-ribbons. • Amyloid fibrils of BCAB and its mutants decrease eukaryotic cells viability. • The fibrils cytotoxicity doesn't depend on their length, secondary structure, and ANS binding. • Specific feature of the low-toxic amyloids is the lateral protofilaments association. [ABSTRACT FROM AUTHOR]

Kinetic investigation on the wild-type apomyoglobin and its 12 mutants with substitutions of hydrophobic residues by Ala was performed using stopped-flow fluorescence. Characteristics of the kinetic intermediate I and the folding nucleus were derived solely from kinetic data, namely, the slow-phase folding rate constants and the burst-phase amplitudes of Trp fluorescence intensity. This allowed us to pioneer the phi-analysis for apomyoglobin. As shown, these mutations drastically destabilized the native state N and produced minor (for conserved residues of G, H helices) or even negligible (for nonconserved residues of B, C, D, E helices) destabilizing effect on the state I. On the other hand, conserved residues of A, G, H helices made a smaller contribution to stability of the folding nucleus at the rate-limiting I-->N transition than nonconserved residues of B, D, E helices. Thus, conserved side chains of the A-, G-, H-residues become involved in the folding nucleus before crossing the main barrier, whereas nonconserved side chains of the B-, D-, E-residues join the nucleus in the course of the I-->N transition., (Copyright 2010 Biophysical Society. Published by Elsevier Inc. All rights reserved.)