ROLE OF QUATERNARY STRUCTURE IN MUSCLE CREATINE-KINASE STABILITY - TRYPTOPHAN-210 IS IMPORTANT FOR DIMER COHESION

A mutant of the dimeric rabbit muscle creatine kinase (MRI-CR) in whic h tryptophan 210 was replaced has been studied to assess the role of t his residue in dimer cohesion and the importance of the dimeric state for the native enzyme stability. Wild-type protein equilibrium unfoldi ng induced by guanidine hydrochloride occurs through intermediate stat es with formation of a molten globule and a premolten globule, Unlike the wild-type enzyme, the mutant inactivates at lower denaturant conce ntration and the loss of enzymatic activity is accompanied by the diss ociation of the dimer into two apparently compact monomers, However, t he Stokes radius of the monomer increases with denaturant concentratio n as determined by size exclusion chromatography, indicating that, upo n monomerization, the protein structure is destabilized. Binding of 8- anilinonaphthalene-1-sulfonate shows that the dissociated monomer expo ses hydrophobic patches at its surface, suggesting that it could be a molten globule, At higher denaturant concentrations, both wild-type an d mutant follow similar denaturation pathways with formation of a prem olten globule around 1.5-M guanidine, indicating that tryptophan 210 d oes not contribute to a large extent to the monomer conformational sta bility, which may be ensured in the dimeric state through quaternary i nteractions. Proteins 32:43-51, 1998, (C) 1998 Wiley-Liss, Inc.