Replica exchange molecular dynamics simulations reveal self-association sites in M-crystallin caused by mutations provide insights of cataract

Crystallins are ubiquitous, however, prevalence is seen in eye lens. Eye lens crystallins are long-lived and structural intactness is required for maintaining lens transparency and protein solubility. Mutations in crystallins often lead to cataract. In this study, we performed mutations at specific sites of M-crystallin, a close homologue of eye lens crystallin and studied by using replica exchange molecular dynamics simulation with generalized Born implicit solvent model. Mutations were made on the Ca2+ binding residues (K34D and S77D) and in the hydrophobic core (W45R) which is known to cause congenital cataract in homologous γD-crystallin. The chosen mutations caused large motion of the N-terminal Greek key, concomitantly broke the interlocking Greek keys interactions and perturbed the compact core resulting in several folded and partially unfolded states. Partially unfolded states exposed large hydrophobic patches that could act as precursors for self-aggregation. Accumulation of such aggregates is the potential cause of cataract in homologous eye lens crystallins.

Replica Exchange Molecular Dynamics Simulations Reveal Self-association Sites in M- Crystallin Caused by Mutations Provide Insights of Cataract

Crystallins are ubiquitous, however, prevalence is seen in eye lens. Eye lens crystallins are long-lived and structural intactness is required for maintaining lens transparency and protein solubility. Mutations in crystallin often lead to cataract. In this study, we performed mutations at specific sites of M-crystallin, a close homologue of eye lens crystallin and studied by employing replica exchange molecular dynamics with generalized Born solvation model. Mutations were made on the Ca2+ binding residues (K34D and S77D) and in the hydrophobic core (W45R) which is known to cause congenital cataract in homologous γD-crystallin. The chosen mutations caused large motion of the N-terminal Greek key, concomitantly break the interlocking Greek keys interactions and perturbed the compact core resulting in several folded and partially unfolded states. Partially unfolded states expose large hydrophobic patches that can act as precursors for self-aggregation. Accumulation of such aggregates is the potential cause of cataract in homologous crystallins.

Mechanism of the small ATP-independent chaperone Spy is substrate specific

ATP-independent chaperones are usually considered to be holdases that rapidly bind to non-native states of substrate proteins and prevent their aggregation. These chaperones are thought to release their substrate proteins prior to their folding. Spy is an ATP-independent chaperone that acts as an aggregation inhibiting holdase but does so by allowing its substrate proteins to fold while they remain continuously chaperone bound, thus acting as a foldase as well. The attributes that allow such dual chaperoning behavior are unclear. Here, we used the topologically complex protein apoflavodoxin to show that the outcome of Spy’s action is substrate specific and depends on its relative affinity for different folding states. Tighter binding of Spy to partially unfolded states of apoflavodoxin limits the possibility of folding while bound, converting Spy to a holdase chaperone. Our results highlight the central role of the substrate in determining the mechanism of chaperone action.

Cytochrome c: A Multifunctional Protein Combining Conformational Rigidity with Flexibility

Cytochrome has served as a model system for studying redox reactions, protein folding, and more recently peroxidase activity induced by partial unfolding on membranes. This review illuminates some important aspects of the research on this biomolecule. The first part summarizes the results of structural analyses of its active site. Owing to heme-protein interactions the heme group is subject to both in-plane and out-of-plane deformations. The unfolding of the protein as discussed in detail in the second part of this review can be induced by changes of pH and temperature and most prominently by the addition of denaturing agents. Both the kinetic and thermodynamic folding and unfolding involve intermediate states with regard to all unfolding conditions. If allowed to sit at alkaline pH (11.5) for a week, the protein does not return to its folding state when the solvent is switched back to neutral pH. It rather adopts a misfolded state that is prone to aggregation via domain swapping. On the surface of cardiolipin containing liposomes, the protein can adopt a variety of partially unfolded states. Apparently, ferricytochrome c can perform biological functions even if it is only partially folded.

Understanding how small helical proteins fold: conformational dynamics of Im proteins relevant to their folding landscapes

Understanding the mechanism of folding of small proteins requires characterization of their starting unfolded states and any partially unfolded states populated during folding. Here, we review what is known from NMR about these states of Im7, a 4-helix bundle protein that folds via an on-pathway intermediate, and show that there is an alignment of non-native structure in urea-unfolded Im7 with the helices of native Im7 that is a consequence of hydrophobic helix-promoting residues also promoting cluster-formation in the unfolded protein. We suggest that this kind of alignment is present in other proteins and is relevant to how native state topology determines folding rates.