Denaturation by guanidinium chloride of dimeric MM-creatine kinase and its proteinase K-nicked form: evidence for a multiple-step process.
Cytosolic MM-creatine kinase is a homodimeric protein. Each monomer can be cleaved by proteinase K at an exposed surface loop, into two fragments K1 and K2, which remain associated. The nicked protein is thus a heterotetrameric protein, named (K1K2)2, made up of two heterodimers K1K2 linked together by their K1 subunit. In non-denaturing conditions, the cleaved protein does not present any measurable difference compared with uncleaved MM-creatine kinase, except for the loss of enzymatic activity. Comparative equilibrium denaturation of the two oligomeric proteins by guanidinium chloride indicates a multistep process with formation of either compact monomer or compact K1K2 dimer, a molten globule and a pre-molten globule state. In the case of the nicked-enzyme, the molten globule is composed of the two peptides K1 and K2, whereas in the pre-molten globule the interactions between K1 and K2 are too weak to maintain their cohesion. At low guanidinium chloride concentration, the proteinase K-nicked protein exhibits a higher accessibility of one of its tryptophan accompanied by a small decrease in its molar ellipticity suggesting a secondary structure loosening of the K1 peptide. Our results suggest that K1 and K2 are not strictly autonomous unfolding units and thus cannot be considered as independent domains.
Clottes
E
E
UPRESA 5013 Centre National de la Recherche Scientifique-Université Lyon I. Bat 3032ème étage, Villeurbanne, France.
Leydier
C
C
Couthon
F
F
Marcillat
O
O
Vial
C
C
D000818:Animals; D002850:Chromatography, Gel; D003402:Creatine Kinase / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism; D003600:Cytosol / Q000201:enzymology; D019281:Dimerization; D019286:Endopeptidase K; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology*; D007527:Isoenzymes; D007700:Kinetics; D046911:Macromolecular Substances; D018482:Muscle, Skeletal / Q000201:enzymology; D011489:Protein Denaturation; D017510:Protein Folding; D011817:Rabbits; D013050:Spectrometry, Fluorescence
D006146:Guanidines; D007527:Isoenzymes; D046911:Macromolecular Substances; D003402:Creatine Kinase; D019286:Endopeptidase K; D019791:Guanidine