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Methods Mol Biol. 2015;1271:159-71. doi: 10.1007/978-1-4939-2330-4_11.

Sequential structural changes in rhodopsin occurring upon photoactivation.

Author information

1
Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, NY, 11794-5215, USA.

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.

PMID:
25697523
DOI:
10.1007/978-1-4939-2330-4_11
[Indexed for MEDLINE]

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