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 ca n be cleaved by proteinase K at an exposed surface loop, into two frag ments K1 and K2, which remain associated. The nicked protein is thus a heterotetrameric protein, named (K1K2)(2). made up of two heterodimer s K1K2 linked together by their K1 subunit. In non-denaturing conditio ns, the cleaved protein does not present any measurable difference com pared with uncleaved MM-creatine kinase, except for the loss of enzyma tic activity. Comparative equilibrium denaturation of the two oligomer ic proteins by guanidinium chloride indicates a multistep process with formation of either compact monomer or compact K1K2 dimer, a molten g lobule and a pre-molten globule state. In the case of the nicked-enzym e, the molten globule is composed of the two peptides K1 and K2, where as in the pre-molten globule the interactions between K1 and K2 are to o weak to maintain their cohesion. At low guanidinium chloride concent ration, the proteinase K-nicked protein exhibits a higher accessibilit y of one of its tryptophan accompanied by a small decrease in its mola r ellipticity suggesting a secondary structure loosening of the K1 pep tide. Our results suggest that K1 and K2 are not strictly autonomous u nfolding units and thus cannot be considered as independent domains.