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 fi rst 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.
CITATION STYLE
Kimata, N., Pope, A., Rashid, D., Reeves, P. J., & Smith, S. O. (2015). Sequential structural changes in rhodopsin occurring upon photoactivation. Methods in Molecular Biology, 1271, 159–171. https://doi.org/10.1007/978-1-4939-2330-4_11
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