G protein-coupled receptors (GPCRs) are a class of membrane proteins that have an essential role in development and function of the endocrine, immune and central nervous systems. The malfunction of certain chemokine GPCRs contribute to the pathology of many diseases including asthma, rheumatioid arthritis, multiple sclerosis, heart disease and metastatic cancer. Consequently the development of drugs to block chemokine receptor function is a major focus in the pharmaceutical industry. Structural information on the GPCR-chemokine complexes is of great importance, but such information is difficult to obtain because of the challenges of producing sufficient quantities of protein to study and the inherent limitations of existing bio-physical methods. In this project we will develop a highly sensitive technology for membrane protein structural analysis using deuterium exchange mass spectrometry (DXMS). While very useful for soluble protein structural analysis, DXMS has not been applied much to membrane proteins because of issues related to sensitivity and the lipid environment needed to preserve membrane protein function. This work will create a microfluidic platform for DXMS studies that will efficiently integrate the various steps in a DXMS experiment. It will establish methods for minimizing the impact of lipid and detergent components on the downstream mass spectral analysis. This will result in a 100-1000 fold increase in sensitivity and extend DXMS methods to any system involving integral membrane proteins. Once developed, the technology will be used to study a number important chemokine receptors and ligands that bind to them.

Public Health Relevance

This work will provide the means to obtain vital structural information on small amounts membrane proteins. Such information is needed for the development of drugs to treat many diseases such as asthma, rheumatoid arthritis, multiple sclerosis, heart disease, and cancer. Consequently, it will have a major effect on improving human health.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
Project #
Application #
Study Section
Enabling Bioanalytical and Biophysical Technologies Study Section (EBT)
Program Officer
Edmonds, Charles G
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
City of Hope/Beckman Research Institute
United States
Zip Code
Marsh, James J; Guan, Henry S; Li, Sheng et al. (2013) Structural insights into fibrinogen dynamics using amide hydrogen/deuterium exchange mass spectrometry. Biochemistry 52:5491-502
Hsu, Yuan-Hao; Bucher, Denis; Cao, Jian et al. (2013) Fluoroketone inhibition of Ca(2+)-independent phospholipase A2 through binding pocket association defined by hydrogen/deuterium exchange and molecular dynamics. J Am Chem Soc 135:1330-7
Hailey, Kendra L; Capraro, Dominique T; Barkho, Sulyman et al. (2013) Allosteric switching of agonist/antagonist activity by a single point mutation in the interluekin-1 receptor antagonist, IL-1Ra. J Mol Biol 425:2382-92
Barkho, Sulyman; Pierce, Levi C T; McGlone, Maria L et al. (2013) Distal loop flexibility of a regulatory domain modulates dynamics and activity of C-terminal SRC kinase (csk). PLoS Comput Biol 9:e1003188
Zhang, Adrianna P P; Bornholdt, Zachary A; Liu, Tong et al. (2012) The ebola virus interferon antagonist VP24 directly binds STAT1 and has a novel, pyramidal fold. PLoS Pathog 8:e1002550
White, Mark A; Li, Sheng; Tsalkova, Tamara et al. (2012) Structural analyses of a constitutively active mutant of exchange protein directly activated by cAMP. PLoS One 7:e49932
Lu, Weiya D; Liu, Tong; Li, Sheng et al. (2012) The prohormone proenkephalin possesses differential conformational features of subdomains revealed by rapid H-D exchange mass spectrometry. Protein Sci 21:178-87
Zhang, Adrianna P P; Abelson, Dafna M; Bornholdt, Zachary A et al. (2012) The ebolavirus VP24 interferon antagonist: know your enemy. Virulence 3:440-5
Bale, Shridhar; Dias, Joao M; Fusco, Marnie L et al. (2012) Structural basis for differential neutralization of ebolaviruses. Viruses 4:447-70
Weinreb, Paul H; Li, Sheng; Gao, Sharon X et al. (2012) Dynamic structural changes are observed upon collagen and metal ion binding to the integrin ?1 I domain. J Biol Chem 287:32897-912

Showing the most recent 10 out of 21 publications