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The ability to control or reverse protein aggregation is vital to the production and formulation of therapeutic proteins and may be the key to prevention of a number of neurodegenerative diseases. Our work incorporates experimental studies and computational treatments aimed at elucidating the molecular mechanisms of aggregation. Our simulation studies include coarse-grained approaches and molecular dynamic studies of a small model peptide. Simulations studies of coarse-grained model oligopeptides have the objective of examining structural motifs and crowding on protein aggregation. The potential function of a multi-chain system is expressed in terms of a generalized Go model for a set of sequences with varying different contents of motifs akin to a-helices and b-sheets. Conformational evolution has been considered by conventional Monte Carlo simulation, and by a variation of the Replica Monte Carlo technique that facilitates barrier crossing in glass-like aggregated systems. Foldability and aggregation propensity are monitored as functions of the extent of secondary structures. Our results indicate that an increased proportion of sheet-like structure facilitates folding of isolated chains, while strongly favoring the formation of misfolded aggregates in multichain systems, in agreement with experimental observations. For our molecular dynamics studies, we have chosen to study a 46-bead/3-flavor model (originally developed by Honeycutt and Thirumalai). The model protein’s relatively small size allows for computational viability, but is large enough to contain various structural elements (3 beta-hairpin turns). We are examining the effects of certain mutations on the protein’s aggregation propensity. Aggregation processes are of second or higher order in protein concentration, and eventually compete with first-order folding kinetics as the expressed protein concentration increases. The cell uses a series of accessory proteins, collectively called molecular chaperones, to help many proteins fold correctly. The objectives of our experimental studies on aggregation are to understand the mechanism of the interaction between molecular chaperones and proteins. We employ one of the best-studied molecular chaperones, the E. coli hsp70 protein DnaK. Hydrophobic interactions have been shown to be important in binding of peptides to DnaK. Certain structural elements, hydrogen bonding, electrostatic interaction and van der Waals forces, are involved in the recognition of polypeptide and protein by molecular chaperone DnaK. We are studying the interaction between DnaK and fluorescein-labeled peptides with fluorescence polarization. DnaK and selected mutants are produced by a recombinant E. coli Top10 strain in our laboratory. Current research » |
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