DETERMINING THE MOLECULAR ENVELOPE OF THE COMPLEX OF A G-PROTEIN AND ITS RECEPTO

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. G-protein coupled receptors are 7-transmembrane helix proteins that mediate the majority of cellular responses to hormones, neurotransmitters, and sensory stimuli. In collaboration with Dr. Brian Kobilka, we have been studying crystal structures of the beta2-adrenergic receptor, a well-characterized G-protein coupled receptor. These were the first structures of a ligand-activated G protein coupled receptor. Thus far all structures of this and related receptors have been of the inactive state, and there are no structures of the active complex of receptor and G protein. A key question is how activating ligands stabilize the conformation of the receptor that can interact with its cognate heterotrimeric G protein and trigger nucleotide exchange. We have recently succeeded in producing a stable complex of the receptor bound to an activating ligand and a G protein. The complex, approximately 150kDa, is monodisperse as determined by size exclusion chromatography and electron microscopy. We propose solution x-ray scattering measurements to assess if we can obtain information about the overall shape of the complex, since we have the crystal structures of the two individual components. The determination of the stoichiometry between the receptor and G protein would be of significant importance as there are a number of conflicting results on oligomerization property of the receptor in the complex. We would also like to apply solution scattering to study effects of ligand and antagonists/agonists in the overall structure of the complex.