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 (MM-CK) in which tryptophan 210 was replaced has been studied to assess the role of this residue in dimer cohesion and the importance of the dimeric state for the native enzyme stability. Wild-type protein equilibrium unfolding induced by guanidine hydrochloride occurs through intermediate states with formation of a molten globule and a premolten globule. Unlike the wild-type enzyme, the mutant inactivates at lower denaturant concentration and the loss of enzymatic activity is accompanied by the dissociation of the dimer into two apparently compact monomers. However, the Stokes radius of the monomer increases with denaturant concentration as determined by size exclusion chromatography, indicating that, upon monomerization, the protein structure is destabilized. Binding of 8-anilinonaphthalene-1-sulfonate shows that the dissociated monomer exposes hydrophobic patches at its surface, suggesting that it could be a molten globule. At higher denaturant concentrations, both wild-type and mutant follow similar denaturation pathways with formation of a premolten globule around 1.5-M guanidine, indicating that tryptophan 210 does not contribute to a large extent to the monomer conformational stability, which may be ensured in the dimeric state through quaternary interactions.
Perraut
C
C
UFR Chimie-Biochimie, Université Claude Bernard Lyon I, Villeurbanne, France.
Clottes
E
E
Leydier
C
C
Vial
C
C
Marcillat
O
O
D000817:Anilino Naphthalenesulfonates / Q000494:pharmacology; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D003402:Creatine Kinase / Q000737:chemistry* / Q000187:drug effects / Q000235:genetics; D019281:Dimerization; D004795:Enzyme Stability; D005453:Fluorescence; D019791:Guanidine / Q000494:pharmacology; D006902:Hydroxymercuribenzoates / Q000494:pharmacology; D007527:Isoenzymes; D016297:Mutagenesis, Site-Directed; D009206:Myocardium / Q000201:enzymology; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D014364:Tryptophan / Q000737:chemistry* / Q000235:genetics
D000817:Anilino Naphthalenesulfonates; D006902:Hydroxymercuribenzoates; D007527:Isoenzymes; C030601:4-hydroxymercuribenzoate; C027132:1-anilino-8-naphthalenesulfonate; D014364:Tryptophan; D003402:Creatine Kinase; D019791:Guanidine