Work in the present grant period developed a new hydrogen exchange method that appears to define three intermediates in the cytochrome c folding pathway. Also kinetic experiments were done to define the barriers that cytochrome c must overcome in their folding trajectory. The information obtained points to a coherent set of steps that may describe the major pathway in cytochrome c folding. The proposed work is designed to test and build on these hypotheses, which can be listed as follows. 1) The unfolded state: Under normal solution conditions, unfolded polypeptides exist as non-specifically contracted chains. In stopped-flow folding experiments that start by diluting unfolded proteins from high denaturant, this biased U condition is reached very rapidly (less than 1 msec), and does not represent a productive folding intermediate. 2) Nucleation: Folding begins with and initial energetically uphill large scale conformational search to reach a transition state nucleus that can support forward folding steps in a downhill manner, and therefore must be native-like in some topological sense. This diffusional search-dependent step typically requires 1 to 10 msec. 3) Folding intermediates: From the folding nucleus, the cyt c chain moves energetically downhill through a sequence of three increasingly native-like, cooperatively structured intermediates to the native state. The step from one intermediate to the next requires only a fast, small scale conformational search. 4) Error barriers: In this downhill sequence the refolding protein may, often with high probability, encounter one or more optional, error-dependent misfold- reorganization barriers that can slow native state acquisition, typically (for small proteins) to the approximate 1 second time scale. These hypotheses will be tested by applying our earlier hydrogen exchange pulse labeling method, our newer native state hydrogen exchange method. 2D NMR, other spectroscopies (CD, fluorescence, absorbance), and rapid reaction methods (stopped-flow). The experiments will use mammalian, bacterial, and genetically engineered versions of cytochrome c as model proteins, and other useful model proteins.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM031847-16
Application #
2838487
Study Section
Molecular and Cellular Biophysics Study Section (BBCA)
Project Start
1983-12-01
Project End
2001-11-30
Budget Start
1998-12-01
Budget End
1999-11-30
Support Year
16
Fiscal Year
1999
Total Cost
Indirect Cost
Name
University of Pennsylvania
Department
Biochemistry
Type
Schools of Medicine
DUNS #
042250712
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
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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