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Protein Science (2004), 13:476-486. Published by Cold Spring Harbor Laboratory Press. Copyright © 2004 The Protein Society
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Hydrogen/deuterium exchange studies of native rabbit MM-CK dynamics

Hortense Mazon1, Olivier Marcillat1, Eric Forest2 and Christian Vial1

1 Unité Mixte de Recherche Centre National de la Recherche Scientifique 5013, Biomembranes et enzymes associés, Université Claude Bernard Lyon I, 69622 Villeurbanne cedex, France
2 Laboratoire de spectrométrie de masse des protéines, Institut de Biologie Structurale, CNRS (Unité Mixte de Recherche 5075)/Centre de l’Energie Atomique/Université Joseph Fourier, 38027 Grenoble cedex, France

(RECEIVED August 19, 2003; FINAL REVISION October 13, 2003; ACCEPTED October 16, 2003)



Abstract

Creatine kinase (CK) isoenzymes catalyse the reversible transfer of a phosphoryl group from ATP onto creatine. This reaction plays a very important role in the regulation of intracellular ATP concentrations in excitable tissues. CK isoenzymes are highly resistant to proteases in native conditions. To appreciate localized backbone dynamics, kinetics of amide hydrogen exchange with deuterium was measured by pulse-labeling the dimeric cytosolic muscle CK isoenzyme. Upon exchange, the protein was digested with pepsin, and the deuterium content of the resulting peptides was determined by liquid chromatography coupled to mass spectrometry (MS). The deuteration kinetics of 47 peptides identified by MS/MS and covering 96% of the CK backbone were analyzed. Four deuteration patterns have been recognized: The less deuterated peptides are located in the saddle-shaped core of CK, whereas most of the highly deuterated peptides are close to the surface and located around the entrance to the active site. Their exchange kinetics are discussed by comparison with the known secondary and tertiary structures of CK with the goal to reveal the conformational dynamics of the protein. Some of the observed dynamic motions may be linked to the conformational changes associated with substrate binding and catalytic mechanism.

Keywords: Creatine kinase dynamics; hydrogen exchange; mass spectrometry

Abbreviations: H/D, hydrogen/deuterium • DTT, dithiothreitol • LC/MS, liquid chromatography/mass spectrometry • MM-CK, cytosolic dimeric creatine kinase MM • MS/MS, tandem mass spectrometry • NH, amide hydrogen • TFA, trifluoroacetic acid

Reprint requests to: Christian Vial, UMR 5013 CNRS, Université Claude Bernard Lyon I, 43 boulevard du 11 Novembre 1918, 69622 Villeurbanne cedex, France; e-mail: christian.vial{at}univ-lyon1.fr; fax: 33-472-431-557.

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.03380604.

Supplemental material: See www.proteinscience.org


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