We have developed innovative methods of structural mass spectrometry based on hydroxyl radical modification of proteins. These structural mass spectrometry methods have recently gained widespread acceptance and are ripe for further development. In this proposal we will: increase the sensitivity of protein footprinting methods ~1000 fold;integrate docking approaches with protein footprinting data to probe the structure of protein complexes and develop methods to examine the dynamics of water in proteins using footprinting. Our preliminary data has shown feasibility for the examination of the G- protein coupled receptor rhodopsin in its ground and photo-activated states using increased x-ray flux density and shorter exposure times. Within Aim 1 we will use increased x-ray flux density to further explore the structural and solvent dynamics accompanying GPCR activation and the structural mechanism of signaling that mediates information to downstream signaling proteins. Our guiding hypothesis is that highly conserved structural motifs that include bound waters are reorganized to provide a highly controlled signaling channel.
In Aim 2 we will further develop a novel O18 water labeling- radiolysis technique to examine the locations and dynamics of structural waters and the exchange properties of bulk water in multiple biological states of interest for rhodopsin and actin.
In Aim 3 we will perfect targeted MS approaches to detect low abundance modifications in protein footprinting experiments to enhance the number of amino acids side chains routinely detected by these experiments.
In Aim 4 we will develop computational methods of docking that incorporate footprinting data in structure determination. Our hypothesis is that use of footprinting data will drive correct selection of the correct structure among competing docking solutions that are of comparable energies. Optimized algorithmic approaches will be used to model complexes of myosin or cofilin with actin and complexes of rhodopsin and transducin.

Public Health Relevance

G-protein coupled receptors are the targets of close to half of all drugs;a better understanding of their activation mechanisms would have a major impact on health and disease. Advanced technologies to examine the structure and dynamics of all proteins are needed in biology and medicine to better correlate structure and function.

Agency
National Institute of Health (NIH)
Institute
National Institute of Biomedical Imaging and Bioengineering (NIBIB)
Type
Research Project (R01)
Project #
1R01EB009688-01A1
Application #
7896398
Study Section
Enabling Bioanalytical and Biophysical Technologies Study Section (EBT)
Program Officer
Mclaughlin, Alan Charles
Project Start
2010-05-10
Project End
2014-02-28
Budget Start
2010-05-10
Budget End
2011-02-28
Support Year
1
Fiscal Year
2010
Total Cost
$282,600
Indirect Cost
Name
Case Western Reserve University
Department
Genetics
Type
Schools of Medicine
DUNS #
077758407
City
Cleveland
State
OH
Country
United States
Zip Code
44106
Huang, Wei; Ravikumar, Krishnakumar M; Chance, Mark R et al. (2015) Quantitative mapping of protein structure by hydroxyl radical footprinting-mediated structural mass spectrometry: a protection factor analysis. Biophys J 108:107-15
Salanga, Catherina L; Dyer, Douglas P; Kiselar, Janna G et al. (2014) Multiple glycosaminoglycan-binding epitopes of monocyte chemoattractant protein-3/CCL7 enable it to function as a non-oligomerizing chemokine. J Biol Chem 289:14896-912
Bohon, Jen; D'Mello, Rhijuta; Ralston, Corie et al. (2014) Synchrotron X-ray footprinting on tour. J Synchrotron Radiat 21:24-31
Gupta, Sayan; Chai, Jin; Cheng, Jie et al. (2014) Visualizing the kinetic power stroke that drives proton-coupled zinc(II) transport. Nature 512:101-4
Wang, Benlian; Tsybovsky, Yaroslav; Palczewski, Krzysztof et al. (2014) Reliable determination of site-specific in vivo protein N-glycosylation based on collision-induced MS/MS and chromatographic retention time. J Am Soc Mass Spectrom 25:729-41
Padayatti, Pius S; Wang, Liwen; Gupta, Sayan et al. (2013) A hybrid structural approach to analyze ligand binding by the serotonin type 4 receptor (5-HT4). Mol Cell Proteomics 12:1259-71
Orban, Tivadar; Jastrzebska, Beata; Gupta, Sayan et al. (2012) Conformational dynamics of activation for the pentameric complex of dimeric G protein-coupled receptor and heterotrimeric G protein. Structure 20:826-40
Wang, Liwen; Cvetkov, Teresa L; Chance, Mark R et al. (2012) Identification of in vivo disulfide conformation of TRPA1 ion channel. J Biol Chem 287:6169-76
Gupta, Sayan; D'Mello, Rhijuta; Chance, Mark R (2012) Structure and dynamics of protein waters revealed by radiolysis and mass spectrometry. Proc Natl Acad Sci U S A 109:14882-7
Wang, Liwen; Chance, Mark R (2011) Structural mass spectrometry of proteins using hydroxyl radical based protein footprinting. Anal Chem 83:7234-41

Showing the most recent 10 out of 16 publications