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.
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.
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|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|
|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|
|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|
|Oztug Durer, Zeynep A; Kamal, J K Amisha; Benchaar, Sabrina et al. (2011) Myosin binding surface on actin probed by hydroxyl radical footprinting and site-directed labels. J Mol Biol 414:204-16|
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