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.

Public Health Relevance

We are combining experimental and computational approaches to produce a predictive understanding of protein folding and dynamics. Our focus has expanded to more complicated systems, including an anti-freeze protein and the plug domain of the TonB-dependent transporters involved in pathogenic bacterial iron sequestration. Tying these studies together is our new molecular dynamics Upside package, which is capable of predicting protein folding, dynamics and structure.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM055694-25
Application #
9938596
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Flicker, Paula F
Project Start
1996-08-01
Project End
2021-05-31
Budget Start
2020-06-01
Budget End
2021-05-31
Support Year
25
Fiscal Year
2020
Total Cost
Indirect Cost
Name
University of Chicago
Department
Biochemistry
Type
Schools of Medicine
DUNS #
005421136
City
Chicago
State
IL
Country
United States
Zip Code
60637
Lian, Huada; Qin, Jian; Freed, Karl F (2018) Dielectric virial expansion of polarizable dipolar spheres. J Chem Phys 149:163332
Wang, Zongan; Jumper, John M; Wang, Sheng et al. (2018) A Membrane Burial Potential with H-Bonds and Applications to Curved Membranes and Fast Simulations. Biophys J 115:1872-1884
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Skinner, John J; Wang, Sheng; Lee, Jiyoung et al. (2017) Conserved salt-bridge competition triggered by phosphorylation regulates the protein interactome. Proc Natl Acad Sci U S A 114:13453-13458
French, Alexander R; Sosnick, Tobin R; Rock, Ronald S (2017) Investigations of human myosin VI targeting using optogenetically controlled cargo loading. Proc Natl Acad Sci U S A 114:E1607-E1616

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