Integral membrane proteins constitute approximately 30% of all genes, making them a very important class of proteins. In addition, more than half of all current drug targets are membrane proteins. This class of proteins is involved in a variety of functions including transport to create electrical and chemical gradients, channel function to enable action potentials, receptors that mediate extracellular signaling, and cell-cell 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. While the rate of structure determinations of soluble proteins has dramatically accelerated over the last decade, that of membrane proteins has lagged far behind, reflecting the technical challenges associated with working with this class of proteins. The primary focus of this grant is to utilize solution NMR approaches to study the structure and function of the integral membrane protein DsbB. The proteins DsbA and DsbB work in concert to mediate the formation of disulfide bonds in the periplasm of E. coli. DsbA is a soluble protein that functions as the primary oxidant for proteins in the periplasm whereas DsbB is an integral membrane protein that reoxidizes DsbA and is itself reoxidized by various quinones. Mutations of DsbA in pathogenic bacteria are avirulent since many virulence components contain disulfide bonds, suggesting this pathway may be a useful target for development of novel antibiotics.
Four Specific Aims using NMR and EPR spectroscopy to characterize the structure and function of DsbB in detergent micelles and in the bilayer are proposed:
Aim 1 : Determination of the solution structure of DsbB, an integral membrane protein essential for disulfide formation in the periplasm of E. coli.
Aim 2 : Structural and functional characterization of the quinone binding site on DsbB.
Aim 3 : Characterization of DsbB dynamics in detergent and in the bilayer.
Aim 4 : Characterization of the DsbB-DsbA interaction. ? ? ?
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|Früh, Virginie; Zhou, Yunpeng; Chen, Dan et al. (2010) Application of fragment-based drug discovery to membrane proteins: identification of ligands of the integral membrane enzyme DsbB. Chem Biol 17:881-91|
|Zhou, Yunpeng; Cierpicki, Tomasz; Jimenez, Ricardo H Flores et al. (2008) NMR solution structure of the integral membrane enzyme DsbB: functional insights into DsbB-catalyzed disulfide bond formation. Mol Cell 31:896-908|