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;G? and G??). 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-G??? signaling complex and the phosphorhodopsin-arrestin1 com- plex. 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 unre- lated complexes.
In Aim 1 : We will identify novel sets of bicelles mixtures that improve the affinity between rhodopsin-transdicin 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 synthe- sis, 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.
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.
|Vishnivetskiy, Sergey A; Zhan, Xuanzhi; Chen, Qiuyan et al. (2014) Arrestin expression in E. coli and purification. Curr Protoc Pharmacol 67:Unit 2.11.|
|Thaker, Tarjani M; Sarwar, Maruf; Preininger, Anita M et al. (2014) A transient interaction between the phosphate binding loop and switch I contributes to the allosteric network between receptor and nucleotide in G?i1. J Biol Chem 289:11331-41|
|Kaya, Ali I; Lokits, Alyssa D; Gilbert, James A et al. (2014) A conserved phenylalanine as a relay between the ?5 helix and the GDP binding region of heterotrimeric Gi protein ? subunit. J Biol Chem 289:24475-87|
|Zhuang, Tiandi; Chen, Qiuyan; Cho, Min-Kyu et al. (2013) Involvement of distinct arrestin-1 elements in binding to different functional forms of rhodopsin. Proc Natl Acad Sci U S A 110:942-7|
|Preininger, Anita M; Meiler, Jens; Hamm, Heidi E (2013) Conformational flexibility and structural dynamics in GPCR-mediated G protein activation: a perspective. J Mol Biol 425:2288-98|
|Hamm, Heidi E; Kaya, Ali I; Gilbert 3rd, James A et al. (2013) Linking receptor activation to changes in Sw I and II of G* proteins. J Struct Biol 184:63-74|
|Vishnivetskiy, Sergey A; Chen, Qiuyan; Palazzo, Maria C et al. (2013) Engineering visual arrestin-1 with special functional characteristics. J Biol Chem 288:3394-405|
|Kaya, Ali I; Thaker, Tarjani M; Preininger, Anita M et al. (2011) Coupling efficiency of rhodopsin and transducin in bicelles. Biochemistry 50:3193-203|