This work joins hydrogen exchange methodology with two dimensional NMR measurement and the capability for site-directed mutagenesis to pursue problems in protein structure and function. The test protein is horse heart cytochrome c. In the first stage of this work, proton resonances in reduced and oxidized cytochrome c were assigned and methods were developed for measurement of the exchange of all the amide NH protons and some side chain protons. This capability will be used to study the fundamental problems in protein chemistry by obtaining proton-resolved H-exchange data on various structural forms and functional states of cytochrome c. Changes in structure and dynamics due to chemical modifications and mutations will be studied by H-exchange and 2D NMR spectroscopy. Measurement of the fast exchanging protons, which are not involved in structure, will help to calibrate electrostatic effects and test current electrostatic theories. Measurement of the slowest hydrogens will be used to study global stability and the issue of remaining structure in globally and locally unfolded protein forms. The broad middle range of exchanging hydrogens will be examined to learn about locally cooperative dynamical motions, stabilizing interactions, and protein design. Experiments in progress with different functional forms of cytochrome c will be continued. H-exchange differences between the reduced and oxidized forms are expected to illuminate the interaction between structural energy and setting of the heme redox potential. H-exchange measurements on cytochrome c will be done while it is complexed to cytochrome c peroxidase. The growing library of site-resolved HX data, at over 100 probe points throughout the cytochrome c structure in various structural and functional states, is expected to reveal the kinds of structural fluctuations that underly the HX process, and help develop the utility of this kind of information for study of structure, dynamics, and energy, and functionally related changes in these parameters.
| 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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