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.
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.
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