Abstract
Hydrogen–deuterium exchange (HDX) is a technique that measures the exchange of protein hydrogens for deuteriums in a D2O-containing buffer, providing readout of the structural dynamics. Histidine hydrogen–deuterium exchange mass spectrometry (His-HDX-MS) is a variation of this technique that measures the slow HDX of imidazole C2 hydrogens of histidines. This measurement, when accompanied by pH titration, provides both pK as and half-lives (t 1/2) of the HDX reaction for individual histidine residues in proteins. The pK a and t 1/2 values indicate the electrostatic environment and the degree of side-chain solvent accessibility of the histidine residues, respectively. Herein we describe an experimental protocol to characterize rhodopsin by His-HDX-MS. This technique can be used to monitor different states of rhodopsin and might be useful for monitoring longtime scale events in other GPCRs.
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Acknowledgments
The work was supported by funding from the Cleveland Foundation and National Eye Institute EY019718.
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Lodowski, D.T., Miyagi, M. (2015). Analysis of Conformational Changes in Rhodopsin by Histidine Hydrogen–Deuterium Exchange. 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_9
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DOI: https://doi.org/10.1007/978-1-4939-2330-4_9
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Online ISBN: 978-1-4939-2330-4
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