title: Sequential structural changes in rhodopsin occurring upon photoactivation.
abstract: We describe the use of solid-state magic angle spinning NMR spectroscopy for characterizing the structure and dynamics of dark, inactive rhodopsin and the active metarhodopsin II intermediate. Solid-state NMR spectroscopy is well suited for structural measurements in both detergent micelles and membrane bilayer environments. We first outline the methods for large-scale production of stable, functional rhodopsin containing (13)C- and (15)N-labeled amino acids. The expression methods make use of eukaryotic HEK293S cell lines that produce correctly folded, fully functional receptors. We subsequently describe the basic methods used for solid-state magic angle spinning NMR measurements of chemical shifts and dipolar couplings, which provide information on rhodopsin structure and dynamics, and describe the use of low-temperature methods to trap the active metarhodopsin II intermediate.
authors.affiliation: Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, NY, 11794-5215, USA.
authors.forename: Naoki
authors.initials: N
authors.lastname: Kimata
authors.forename: Andreyah
authors.initials: A
authors.lastname: Pope
authors.forename: Dawood
authors.initials: D
authors.lastname: Rashid
authors.forename: Philip J
authors.initials: PJ
authors.lastname: Reeves
authors.forename: Steven O
authors.initials: SO
authors.lastname: Smith
mesh_terms: D000596:Amino Acids / Q000737:chemistry; D002460:Cell Line; D006801:Humans; D008051:Lipid Bilayers / Q000737:chemistry; D019906:Nuclear Magnetic Resonance, Biomolecular; D010777:Photochemistry; D017433:Protein Structure, Secondary; D012243:Rhodopsin / Q000737:chemistry*
chemical_list: D000596:Amino Acids; D008051:Lipid Bilayers; C013110:metarhodopsins; D012243:Rhodopsin