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 13C- and 15N-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.
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Acknowledgments
This work was supported by NIH-NSF instrumentation grants (S10 RR13889 and DBI-9977553), a grant from the NIH to S. O. S (GM-41412). We gratefully acknowledge the W.M. Keck Foundation for support of the NMR facilities in the Center of Structural Biology at Stony Brook.
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Kimata, N., Pope, A., Rashid, D., Reeves, P.J., Smith, S.O. (2015). Sequential Structural Changes in Rhodopsin Occurring upon Photoactivation. In: Jastrzebska, B. (eds) Rhodopsin. Methods in Molecular Biology, vol 1271. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2330-4_11
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DOI: https://doi.org/10.1007/978-1-4939-2330-4_11
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