Solution NMR Structures of Integral Membrane Proteins

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DESCRIPTION (provided by applicant): Integral membrane proteins comprise ca. 30% of all genes and are the targets of the vast majority of current drugs. This class of proteins is involved in a variety of critical functions including transport to create electrical and chemical gradients, channel function to enable action potentials, receptors that mediate extracellular signaling, and call-call recognition. The critical role of these proteins is further reflected in the identification of mutations in various membrane proteins leading to disease sates such as the CFTR in cystic fibrosis, rhodopsin in retinitis pigmentosa, and the insulin receptor in diabetes. In contrast to the dramatic acceleration in the rate of structure determinations of soluble proteins, there is a relative paucity of structural data on membrane proteins with only approximately 30 structures in the PDB. Recant successes in the application of solution NMR methodology to the structure determination of membrane proteins, including our own determination of the structure of OmpA, suggest solution NMR can can play a significant role in this area. Aim 1 proposes to develop a generalizable approach to the application of solution NMR to the determination of high-resolution backbone structures of membrane proteins, particularly helical membrane proteins. We propose to employ the methods we used for OmpA with the addition of long-range distance restraints from election - nuclear relaxation and refinement with dipolar couplings. We are testing various approaches to each of these, including development of appropriate media to achieve alignment, using OmpA as a test case. Aim 2 proposes to apply these methods to the 21kDa helical membrane protein DsbB. As a purportedly monomeric small helical membrane protein, this is the perfect test for the methods developed in Aim 1. DsbB is a member of the Dsb family of proteins, all of which are essential for proper disulfide bond formation in the periplasm of E. coli. Mutation of DsbB homologs in pathogenic bacteria render them avirulent due to the improper disulfide formation in many proteins that are essential for virulence, thus making this a potential antibacterial target. DsbB oxidizes the periplasmic protein DsbA by means of two redox-active dithiol/disulfide sites, subsequently transferring these reducing equivalents to ubiquinone or menaquinone. A structure of this protein is critical to understand recognition of both DsbA and quinones, and the catalytic mechanism.