Rhodopsin pp 123-132 | Cite as

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

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

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.

Key words

Histidine Hydrogen–deuterium exchange Mass spectrometry pKa Solvent accessibility 

Notes

Acknowledgments

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

References

  1. 1.
    Dempsey CE (2001) Hydrogen exchange in peptides and proteins using NMR-spectroscopy. Prog Nucl Magn Reson Spectrosc 39:135–170CrossRefGoogle Scholar
  2. 2.
    Harris TM, Randall JC (1965) Deuterium exchange reactions at the C2-position of imidazoles. Chem Ind 41:1728–1729PubMedGoogle Scholar
  3. 3.
    Amyes TL, Diver ST, Richard JP et al (2004) Formation and stability of N-heterocyclic carbenes in water: the carbon acid pKa of imidazolium cations in aqueous solution. J Am Chem Soc 126:4366–4374CrossRefPubMedGoogle Scholar
  4. 4.
    Vaughan JD, Mughrabi ZE, Wu C (1970) The kinetics of deuteration of imidazole. J Org Chem 35:1141–1145CrossRefGoogle Scholar
  5. 5.
    Miyagi M, Nakazawa T (2008) Determination of pKa values of individual histidine residues in proteins using mass spectrometry. Anal Chem 80:6481–6487CrossRefPubMedGoogle Scholar
  6. 6.
    Mullangi V, Zhou X, Ball DW et al (2012) Quantitative measurement of solvent accessibility of histidine imidazole groups in proteins. Biochemistry 51:7202–7208CrossRefPubMedCentralPubMedGoogle Scholar
  7. 7.
    Miyagi M, Wan Q, Ahmad MF et al (2011) Histidine hydrogen-deuterium exchange mass spectrometry for probing the microenvironment of histidine residues in dihydrofolate reductase. PLoS One 6:e17055CrossRefPubMedCentralPubMedGoogle Scholar
  8. 8.
    Wimalasena DS, Janowiak BE, Lovell S et al (2010) Evidence that histidine protonation of receptor-bound anthrax protective antigen is a trigger for pore formation. Biochemistry 49:6973–6983CrossRefPubMedCentralPubMedGoogle Scholar
  9. 9.
    Lodowski DT, Palczewski K, Miyagi M (2010) Conformational changes in the G protein-coupled receptor rhodopsin revealed by histidine hydrogen-deuterium exchange. Biochemistry 49:9425–9427CrossRefPubMedCentralPubMedGoogle Scholar
  10. 10.
    Dong J, Callahan KL, Borotto NB et al (2014) Identifying Zn-bound histidine residues in metalloproteins using hydrogen-deuterium exchange mass spectrometry. Anal Chem 86:766–773CrossRefPubMedCentralPubMedGoogle Scholar
  11. 11.
    Hirs CHW (1967) Performic acid oxidation. Methods Enzymol 11:197–199CrossRefGoogle Scholar

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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