Stabilization of Membrane Protein Signaling Complexes

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DESCRIPTION (provided by applicant): Determination of the structures of complexes between membrane proteins and their signaling partners is a pressing problem that has been hindered by the lack of methods for long-term stabilization of the complexes. We will use the visual signal transduction pathway as a model system to develop stabilization strategies since an optical readout can be used to easily monitor complex stability. Visual signal transduction depends upon the GPCR rhodopsin and heterotrimeric guanine nucleotide binding proteins (G-proteins; G1 and G23). Structural characterization of opsin, rhodopsin, and G-proteins in multiple states has provided exceptional insight into the basic mechanisms of visual signaling. However, the molecular understanding of the G protein signaling cycle is far from complete since the details of the complexes formed between signaling molecules are not known. Our targets will be stabilization of the rhodopsin-G123 signaling complex and the phosphorhodopsin-arrestin1 complex. These studies aim to identify what factors are important for the stabilization of biologically transient transmembrane signaling complexes, which will reveal general principles important for the stabilization of unrelated complexes. In Aim 1 we will identify novel sets of bicelles mixtures that improve the affinity between rhodopsin-transducin and rhodopsin-arrestin1. Once we have identified bicelles mixtures that improve the coupling efficiency and half-life of the complex, we will subject these to crystallization trials. In Aim 2 we will use standard and novel peptide detergents with negatively-charged or phosphorylated head groups as agents to stabilize the rhodopsin-transducin and rhodopsin-arrestin1 complexes. Following synthesis, we will monitor the stability of each complex using peptide detergents in isolation or in combination with micellar detergents to evaluate their efficacy in stabilization of membrane protein complexes. In Aim 3 we will evaluate the effects of modification of transducin and arrestin1 on the affinity of each com- plex. Altered proteins with improved affinity will be evaluated structurally. PUBLIC HEALTH RELEVANCE: We are working to improve the methods for structural investigation of the transient signaling complexes using complexes of rhodopsin as a model system. This proposal focuses on investigating solubilization reagents for membrane proteins that mimic biological membranes and improve complex affinity.