Rhodopsin pp 123-132 | Cite as

Analysis of Conformational Changes in Rhodopsin by Histidine Hydrogen–Deuterium Exchange

Part of the Methods in Molecular Biology book series (MIMB, volume 1271)


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.

Key words

Histidine Hydrogen–deuterium exchange Mass spectrometry pKa Solvent accessibility 



The work was supported by funding from the Cleveland Foundation and National Eye Institute EY019718.


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Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  1. 1.Case Center for Proteomics and BioinformaticsCase Western Reserve UniversityClevelandUSA
  2. 2.Department of Pharmacology Case Western Reserve UniversityCase Western Reserve UniversityClevelandUSA

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