Denaturant sensitive regions in creatine kinase identified by hydrogen/deuterium exchange.
The GdmHCl-induced unfolding of creatine kinase (CK) has been studied by hydrogen/deuterium (H/D) exchange combined with mass spectrometry. MM-CK unfolded for various periods in different denaturant concentrations was pulsed-labeled with deuterium to identify different conformational intermediate states. For all denaturation times or GdmHCl concentrations, we observed variable proportions of only two species. The low-mass envelope of isotope peaks corresponds to a species that has gained about 10 deuteriums more than native CK, and the high-mass envelope to a completely deuterated species. To localize precisely the unfolded regions in the states highly populated during denaturation, the protein was digested with two proteases (pepsin and type XIII protease) after H/D exchange and rapid quenching of the reaction. The two sets of fragments obtained were analyzed by liquid chromatography coupled to mass spectrometry to determine the deuterium level in each fragment. Bimodal distributions of deuterium were found for most peptides, indicating that these regions were either folded or unfolded. This behavior is consistent with cooperative, localized unfolding. However, we observed a monomodal distribution of deuterium in two regions (1-12 and 162-186). We conclude that the increment of mass observed in the low-mass species of the intact protein (+10 Da) has its origin in these two segments. These regions, which are very sensitive to low GdmHCl concentrations, are involved in the monomer-monomer interface of CK and their perturbation is likely to weaken the dimeric structure. At higher denaturant concentration, this would induce dissociation of the dimer.
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; D000818:Animals; D003402:Creatine Kinase / Q000737:chemistry* / Q000378:metabolism*; D041961:Deuterium Exchange Measurement; D013058:Mass Spectrometry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011489:Protein Denaturation; D011817:Rabbits; D021241:Spectrometry, Mass, Electrospray Ionization
D003402:Creatine Kinase