ARTICLEHydrogen/deuterium exchange studies of native rabbit MM-CK dynamics |
| Hortense Mazon 1, Olivier Marcillat 1, Eric Forest 2, Christian Vial 1 * |
1Unité Mixte de Recherche Centre National de la Recherche Scientifique 5013, Biomembranes et enzymes associés, Université Claude Bernard Lyon I, 69622 Villeurbanne cedex, France
2Laboratoire 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
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| email: Christian Vial (christian.vial@univ-lyon1.fr) |
*Correspondence to Christian Vial, UMR 5013 CNRS, Université Claude Bernard Lyon I, 43 boulevard du 11 Novembre 1918, 69622 Villeurbanne cedex, France; fax: 33-472-431-557.
| Creatine kinase dynamics hydrogen exchange mass spectrometry |
| Proteins | 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 |
| 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. |
Received: 19 August 2003; Revised: 13 October 2003; Accepted: 16 October 2003
10.1110/ps.03380604
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