The major challenge for the protein chemist is to explain how proteins implement their limited repertoire of structural and biophysical properties to produce their myriad functions. Unlike any other laboratory method, hydrogen exchange (HX) behavior encodes detailed quantitative information, at amino acid resolution, on the biophysical factors that produce protein function -- structure, structure change, interactions, dynamics, and energetics. Previous versions of this NIH grant have been instrumental in developing the HX field by using a variety of NMR methods, but routine NMR analysis is limited to small highly soluble proteins in substantial quantity and high concentration. A developing hydrogen exchange - mass spectrometry technology (HX MS) promises to extend this proven capability to the larger and more complex protein systems that make biology work while requiring only picomoles of protein at sub-micromolar concentrations. This application proposes to perfect the HX MS technology and apply it in studies of important protein systems. They are: 1) apolipoproteins E3 and E4, when lipid- free and lipid-bound, important for cardiovascular and Alzheimer's diseases;2) the definition of autoimmune antibody epitopes in acquired TTP disease, relevant to antibody therapeutics and to protein interactions more generally;3) the Hsp104 protein disaggregase, relevant to the control of protein aggregation and amyloid processing. The full development of HX MS and its demonstration with difficult but centrally important protein systems will provide a uniquely powerful methodology that is widely applicable to the study of protein structure - function problems.

Public Health Relevance

This proposal is directed at the development and use of a uniquely powerful technology, hydrogen exchange measured by mass spectrometry, for studying the proteins that make biology work. The technology will be used to study three carefully chosen protein systems that are key players in biological function and dysfunction. These are lipid metabolism important in cardiovascular and Alzheimer's disease, antibody interactions important in autoimmunity diseases, and protein aggregation important in amyloid formation and the aging process.

Agency
National Institute of Health (NIH)
Type
Research Project (R01)
Project #
2R01GM031847-31
Application #
8755781
Study Section
Macromolecular Structure and Function B Study Section (MSFB)
Program Officer
Wehrle, Janna P
Project Start
Project End
Budget Start
Budget End
Support Year
31
Fiscal Year
2014
Total Cost
Indirect Cost
Name
University of Pennsylvania
Department
Biochemistry
Type
Schools of Medicine
DUNS #
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
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
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
Walters, Benjamin T; Mayne, Leland; Hinshaw, James R et al. (2013) Folding of a large protein at high structural resolution. Proc Natl Acad Sci U S A 110:18898-903
Kan, Zhong-Yuan; Walters, Benjamin T; Mayne, Leland et al. (2013) Protein hydrogen exchange at residue resolution by proteolytic fragmentation mass spectrometry analysis. Proc Natl Acad Sci U S A 110:16438-43
Hu, Wenbing; Walters, Benjamin T; Kan, Zhong-Yuan et al. (2013) Stepwise protein folding at near amino acid resolution by hydrogen exchange and mass spectrometry. Proc Natl Acad Sci U S A 110:7684-9
Walters, Benjamin T; Ricciuti, Alec; Mayne, Leland et al. (2012) Minimizing back exchange in the hydrogen exchange-mass spectrometry experiment. J Am Soc Mass Spectrom 23:2132-9
Mayne, Leland; Kan, Zhong-Yuan; Chetty, Palaniappan Sevugan et al. (2011) Many overlapping peptides for protein hydrogen exchange experiments by the fragment separation-mass spectrometry method. J Am Soc Mass Spectrom 22:1898-905
Kan, Zhong-Yuan; Mayne, Leland; Chetty, Palaniappan Sevugan et al. (2011) ExMS: data analysis for HX-MS experiments. J Am Soc Mass Spectrom 22:1906-15

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