Denaturation by guanidinium chloride of dimeric MM-creatine kinase and its proteinase K-nicked form: Evidence for a multiple-step process

被引:25
作者
Clottes, E [1 ]
Leydier, C [1 ]
Couthon, F [1 ]
Marcillat, O [1 ]
Vial, C [1 ]
机构
[1] UNIV LYON 1,UPRESA 5013 CNRS,F-69622 VILLEURBANNE,FRANCE
来源
BIOCHIMICA ET BIOPHYSICA ACTA-PROTEIN STRUCTURE AND MOLECULAR ENZYMOLOGY | 1997年 / 1338卷 / 01期
关键词
denaturation intermediate state; intrinsic fluorescence MM-creatine kinase; protein domain; molten globule; pre-molten globule;
D O I
10.1016/S0167-4838(96)00186-0
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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.
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页码:37 / 46
页数:10
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