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Understanding the molecular determinants of fibrillogenesis by studying the aggregation propensities of high homologous proteins with different folding pathways., 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 Ml153–149 and zinc-lacking Ml452–151. The results indicate that the three proteins significantly differ in terms of stability and (un)folding mechanisms. Particularly, Ros87 and Ml153–149 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 Ml452–151 folds according to a classic two-state model. Successively, we have monitored the capabilities of Ros87, Ml452–151 and Ml153–149 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 Ml153–149 has formed only amorphous aggregates and Ml452–151 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.

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, the two zinc binding Ros87, Ml1 53>149 and the 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 much more stable at guanidinium denaturation and characterized by folding mechanisms including the presence of an intermediate. On the other hand, the metal lacking Ml4 52 −151 folds according to a classic two>state model. Successively, we have monitored Ros87, Ml4 52 −151 and Ml1 53>149 capabilities to form amyloid fibrils under native conditions. Particularly, we show, by CD, fluorescence, DLS, TEM and SEM experiments, that after 168 hours Ros87 amyloid formation 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, sheding light onto the role of metal ions in the development of protein deposition diseases. 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 Ml153-149 and zinc-lacking Ml452-151. The results indicate that the three proteins significantly differ in terms of stability and (un)folding mechanisms. Particularly, Ros87 and Ml153-149 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 Ml452-151 folds according to a classic two-state model. Successively, we have monitored the capabilities of Ros87, Ml452-151 and Ml153-149 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 Ml153-149 has formed only amorphous aggregates and Ml452-151 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.

Co(II) electronic configuration allows its use as a spectroscopic probe in UV-Vis experiments to characterize the metal coordination sphere that is an essential component of the functional structure of zinc-binding proteins and to evaluate the metal ion affinities of these proteins. Here, exploiting the capability of the prokaryotic zinc finger to use different combinations of residues to properly coordinate the structural metal ion, we provide the UV-Vis characterization of Co(II) addition to Ros87 and its mutant Ros87_C27D which bears an unusual CysAspHis2coordination sphere. Zinc finger sites containing only one cysteine have been infrequently characterized. We show for the CysAspHis2coordination an intensed-dtransition band, blue-shifted with respect to the Cys2His2sphere. These data complemented by NMR and CD data demonstrate that the tetrahedral geometry of the metal site is retained also in the case of a single-cysteine coordination sphere. Co(II) electronic configuration allows its use as a spectroscopic probe in UV-Vis experiments to characterize the metal coordination sphere that is an essential component of the functional structure of zinc-binding proteins and to evaluate the metal ion affinities of these proteins. Here, exploiting the capability of the prokaryotic zinc finger to use different combinations of residues to properly coordinate the structural metal ion, we provide the UV-Vis characterization of Co(II) addition to Ros87 and its mutant Ros87_C27D which bears an unusual CysAspHis(2) coordination sphere. Zinc finger sites containing only one cysteine have been infrequently characterized. We show for the CysAspHis(2) coordination an intense d-d transition band, blue-shifted with respect to the Cys(2)His(2) sphere. These data complemented by NMR and CD data demonstrate that the tetrahedral geometry of the metal site is retained also in the case of a single-cysteine coordination sphere.