Local dynamics measured by hydrogen/deuterium exchange and mass spectrometry of creatine kinase digested by two proteases.
Hydrogen/deuterium exchange coupled to mass spectrometry has been used to investigate the structure and dynamics of native dimeric cytosolic muscle creatine kinase. The protein was incubated in D2O for various time. After H/D exchange and rapid quenching of the reaction, the partially deuterated protein was cleaved in parallel by two different proteases (pepsin or type XIII protease from Aspergillus saitoi) to increase the sequence coverage and spatial resolution of deuterium incorporation. The resulting peptides were analyzed by liquid chromatography coupled to mass spectrometry. In comparison with the 3D structure of MM-CK, the analysis of the two independent proteolysis deuteration patterns allowed us to get new insights into CK local dynamics as compared to a previous study using pepsin [Mazon et al. Protein Science 13 (2004) 476-486]. In particular, we obtained more information on the kinetics and extent of deuterium exchange in the N- and C-terminal extremities represented by the 1-22 and 362-380 pepsin peptides. Indeed, we observed a very different behaviour of the 1-12 and 13-22 type XIII protease peptides, and similarly for the 362-373 and 374-380 peptides. Moreover, comparison of the deuteration patterns of type XIII protease segments of the large 90-126 pepsin peptide led us to identify a small relatively dynamic region (108-114).
Mazon
Hortense
H
UMR CNRS 5013, Biomembranes et enzymes associés, Université Claude Bernard Lyon I, 43, boulevard du 11 novembre 1918, 69622 Villeurbanne cedex, France.
Marcillat
Olivier
O
Forest
Eric
E
Vial
Christian
C
D000595:Amino Acid Sequence; D001230:Aspergillus / Q000201:enzymology; D052283:Creatine Kinase, MM Form / Q000737:chemistry* / Q000378:metabolism*; D003903:Deuterium; D017666:Deuterium Oxide / Q000378:metabolism; D006859:Hydrogen; D007700:Kinetics; D013058:Mass Spectrometry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010434:Pepsin A / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry; D010447:Peptide Hydrolases / Q000378:metabolism*; D017433:Protein Structure, Secondary
D010446:Peptide Fragments; D006859:Hydrogen; D003903:Deuterium; D052283:Creatine Kinase, MM Form; D010447:Peptide Hydrolases; D010434:Pepsin A; D017666:Deuterium Oxide