Project Summary/Abstract Protein folding and dynamics are integral to many biological activities, including chaperone action, degradation, amyloid diseases and aging. Our goal is to combine experimental and computational studies to produce a predictive understanding of protein dynamics through the development of methods capable of simulating folding and dynamics just using physio-chemical principles. Our past studies have focused on single domain proteins. Our efforts have expanded to more complicated systems including the snow flea anti-freeze protein (sfAFP) and TonB-dependent transporters (TBDT) that are relevant to iron sequestration in pathogenic bacteria. Tying these studies together is our new molecular dynamics (MD) package, Upside, which can reversibly fold some proteins up to 97 AA to under 4 C?-RMSD in cpu-days without the use of fragments, homology or evolution. Upside utilizes a number of unique features, including rapid side chain packing that enables simulations using only 3 backbone atoms while retaining considerable detail and avoiding side chain ?rattling?, which slows all-atom methods. We will improve Upside and implement enhanced sampling methods to increase our accuracy and size range, and study protein dynamics as monitored by hydrogen exchange. sfAFP's unique structure challenges conventional wisdom regarding cooperative folding and stability. Lacking a hydrophobic core to promote folding, other factors must contribute to sfAFP's stability. We will test our quantum calculations that sfAFP's H-bonds are unusually stable by measuring amide H/D fractionation factors and NMR J-couplings. We will evaluate whether intrinsic biases in backbone dihedral angles for the PP2 basin in the unfolded state are another major stabilizing factor. This information will be used to improve the Upside simulations. Finally, we will apply our standard folding tools to characterize the folding pathway and compare it to the behavior we expect based on principles derived from proteins with hydrophobic cores. Many aspects of the transport cycle in TBDT remain unknown despite protracted study, including the conformational rearrangement of the plug domain during transport. We will provide the first structure of the plug domain outside the barrel, and so answer whether this structure matches the crystal structure in the barrel. Additionally, the study of Nakamoto's V10C-S120C variant of the BtuB plug enables the investigation of a possible folding or transport intermediate. We will characterize the plug's dynamics while it is in the barrel using HX to observe possible transport-competent states. The mechanism of plug folding and insertion into the barrel will be investigated, with comparative studies for FhuA, a TBDT whose plug domain is intrinsically disordered in solution. These studies represent an exciting combination of protein folding and function, at the interface between soluble and membrane folding, using experiments and complementary folding simulations.
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