My goal is to investigate the structural basis of retinal and neurological disorders using x-ray crystallography. In order to become an independent investigator in the field of structural biology, I propose to undertake a course of training with preeminent x-ray crystallographer, Wayne A. Hendrickson, Department of Biochemistry and Molecular Biophysics, Columbia University.
The specific aims of this proposal concern the structural determination of three proteins that are related to retinal dystrophies: alpha-tocopherol transfer protein (ATTP), cellular retinaldehyde binding protein (CRALBP), and rhodopsin. Mutations in the gene encoding ATTP have been shown to cause a late onset retinal dystrophy and ataxia with isolated vitamin E deficiency, an autosomal recessive condition characterized by a progressive spinocerebellar degeneration. Mutations in CRALBP have been shown to cause a number of autosomal recessive retinal dystrophies. Finally, mutations in rhodopsin have been shown to cause autosomal dominant retinitis pigmentosa (ADRP) and congenital stationary night blindness. ATTP and CRALBP are both cytosolic proteins that bind insoluble hydrophobic small molecules, and regulate their availability to the whole body (for vitamin E) or the photoreceptor (for 11-cis-retinal). The structures of these proteins should help to elucidate the function of this class of proteins. Rhodopsin is a G protein coupled receptor (GPCR), perhaps the most important class of receptor molecules. Although a high resolution structure of ground-state rhodopsin was recently reported, the structure of the activated form of rhodopsin is still unknown.
My aim i s to determine the structure of activated rhodopsin in a complex with transducin, its cognate G protein. Such a structure would have ramifications for the entire GPCR family, as well as disease causing mutations of rhodopsin.
Specific Aim 1 is expression and purification of CRALBP and ATTP.
Specific Aim 2 is crystallization and structure determination of CRALBP and ATTP.
Specific Aim 3 is crystallization and structural determination of native bovine rhodopsin.
Specific Aim 4 is crystallization and structure determination of activated bovine rhodopsin alone or with transducin.
Specific Aim 5 is development of an expression system for rhodopsin suitable for use in Specific Aim 4 and 5, with mutants of rhodopsin as an alternative strategy to pursue the structure of the active state of rhodopsin.
Specific Aim 6 is the use of the systems described in Aims 3, 4, and 5, to analyze the mechanism of signal transduction by rhodopsin using mutants and x-ray structure determination. ? ?
| Min, K Christopher; Jin, Yan; Hendrickson, Wayne A (2011) Large-scale production of a disulfide-stabilized constitutively active mutant opsin. Protein Expr Purif 75:236-41 |
| Janz, Jay M; Sakmar, Thomas P; Min, K Christopher (2007) A novel interaction between atrophin-interacting protein 4 and beta-p21-activated kinase-interactive exchange factor is mediated by an SH3 domain. J Biol Chem 282:28893-903 |
