Folding mechanisms steer the amyloid fibril formation propensity of highly homologous proteins

Abstract

Significant advances in the understanding of the molecular determinants of fibrillogenesis can be expected from comparative studies of the aggregation propensities of proteins with highly homologous structures but different folding pathways. Here, we fully characterize, by means of stopped-flow, T-jump, CD and DSC experiments, the unfolding mechanisms of three highly homologous proteins, zinc binding Ros87 and Ml1_53–149 and zinc-lacking Ml4_52–151. The results indicate that the three proteins significantly differ in terms of stability and (un)folding mechanisms. Particularly, Ros87 and Ml1_53–149 appear to be much more stable to guanidine denaturation and are characterized by folding mechanisms including the presence of an intermediate. On the other hand, metal lacking Ml4_52–151 folds according to a classic two-state model. Successively, we have monitored the capabilities of Ros87, Ml4_52–151 and Ml1_53–149 to form amyloid fibrils under native conditions. Particularly, we show, by CD, fluorescence, DLS, TEM and SEM experiments, that after 168 hours, amyloid formation of Ros87 has started, while Ml1_53–149 has formed only amorphous aggregates and Ml4_52–151 is still monomeric in solution. This study shows how metal binding can influence protein folding pathways and thereby control conformational accessibility to aggregation-prone states, which in turn changes aggregation kinetics, shedding light on the role of metal ions in the development of protein deposition diseases.