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 togethe...

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Bibliographic Details
Published in:Biochimica et biophysica acta 1997-03, Vol.1338 (1), p.37-46
Main Authors: Clottes, Eric, Leydier, Chantal, Couthon, Fabienne, Marcillat, Olivier, Vial, Christian
Format: Article
Language:English
Subjects:
Animals
Chromatography, Gel
Creatine Kinase - chemistry
Creatine Kinase - drug effects
Creatine Kinase - metabolism
Cytosol - enzymology
Denaturation intermediate state
Dimerization
Endopeptidase K
Guanidine
Guanidines - pharmacology
Intrinsic fluorescence
Isoenzymes
Kinetics
Macromolecular Substances
MM-creatine kinase
Molten globule
Muscle, Skeletal - enzymology
Pre-molten globule
Protein Denaturation
Protein domain
Protein Folding
Rabbits
Spectrometry, Fluorescence
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Summary: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.
ISSN:0167-4838
0006-3002
1879-2588
DOI:10.1016/S0167-4838(96)00186-0