This work will use hydrogen exchange (HX), nuclear magnetic resonance (NMR), site-directed mutagenesis, theoretical calculations, and other physical and chemical approaches to study problems in protein structure, dynamics, and function. Projects already begun are directed at the structural bases of HX behavior, kinetic protein folding, equilibrium folding intermediates and protein electrostatics. The structural mechanisms that underlie protein HX behavior will be studied. Local scale effects are being studied using site-directed mutations in recombinant rat cyt c. These results will also quantitatively evaluate the contribution of individual amino acid interactions to protein structural stabilization. To study larger scale effects, we have gathered HX data on cyt c in different functional and chemically modified forms and on cyt c complexed with four different proteins. The detailed H- exchange patterns recorded in this work and in available published work on protein and polypeptide systems will be analyzed. In these analyses the structural contribution to H-exchange will be more clearly displayed by our new ability to remove residue- specific chemical variables. Protein folding experiments are planned to identify the barriers that inhibit cyt c folding and lead to folding heterogeneity. These residue- dependent barriers will be removed by mutagenesis and/or by chemical manipulations already worked out. Fast folding will be studied in the resulting constructs. We will then attempt to populate and study folding intermediates that are otherwise kinetically invisible by mutagenically reinserting new barriers. To study the rate-limiting step in folding, we will use as starting material equilibrium folding intermediates of cyt c that we have characterized previously to be in different stages of unfolding. In this work special approaches will be used to distinguish the time-resolved appearance of native vs. pre-native vs. pre-native forms. An NMR-detected HX method will be used to obtain a site-resolved map of the electrostatic potential around the surface of the highly charged cyt c molecule. The field distribution will be further manipulated by site- directed residue changes. These results will be used in concert with available theoretical models of protein electrostatics to improve the models and also to help understand the role that electrostatics plays in modifying protein HX behavior. Work will be done to determine the reality of a partially structured equilibrium folding intermediate of cyt c that we proposed in previous work.
| Ye, Xiang; Mayne, Leland; Kan, Zhong-Yuan et al. (2018) Folding of maltose binding protein outside of and in GroEL. Proc Natl Acad Sci U S A 115:519-524 |
| Nguyen, David; Mayne, Leland; Phillips, Michael C et al. (2018) Reference Parameters for Protein Hydrogen Exchange Rates. J Am Soc Mass Spectrom 29:1936-1939 |
| Tischer, Alexander; Machha, Venkata R; Frontroth, Juan P et al. (2017) Enhanced Local Disorder in a Clinically Elusive von Willebrand Factor Provokes High-Affinity Platelet Clumping. J Mol Biol 429:2161-2177 |
| Chetty, Palaniappan S; Mayne, Leland; Lund-Katz, Sissel et al. (2017) Helical structure, stability, and dynamics in human apolipoprotein E3 and E4 by hydrogen exchange and mass spectrometry. Proc Natl Acad Sci U S A 114:968-973 |
| Mayne, Leland (2016) Hydrogen Exchange Mass Spectrometry. Methods Enzymol 566:335-56 |
| Hu, Wenbing; Kan, Zhong-Yuan; Mayne, Leland et al. (2016) Cytochrome c folds through foldon-dependent native-like intermediates in an ordered pathway. Proc Natl Acad Sci U S A 113:3809-14 |
| Englander, S Walter; Mayne, Leland; Kan, Zhong-Yuan et al. (2016) Protein Folding-How and Why: By Hydrogen Exchange, Fragment Separation, and Mass Spectrometry. Annu Rev Biophys 45:135-52 |
| Casina, Veronica C; Hu, Wenbing; Mao, Jian-Hua et al. (2015) High-resolution epitope mapping by HX MS reveals the pathogenic mechanism and a possible therapy for autoimmune TTP syndrome. Proc Natl Acad Sci U S A 112:9620-5 |
| Englander, S Walter; Mayne, Leland (2014) The nature of protein folding pathways. Proc Natl Acad Sci U S A 111:15873-80 |
| Chetty, Palaniappan Sevugan; Nguyen, David; Nickel, Margaret et al. (2013) Comparison of apoA-I helical structure and stability in discoidal and spherical HDL particles by HX and mass spectrometry. J Lipid Res 54:1589-97 |
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