Role of C-terminal sequences in the folding of muscle creatine kinase.
Proteinase K selectively nicks the native homodimeric muscle creatine kinase (MM-CK) into two 37.1 kDa N-terminal (K1) and two 5.8 kDa C-terminal (K2) fragments that remain firmly associated in a native-like, although inactive, heterotetrameric structure. This truncated protein has been named (K1K2)(2). To analyze the role of the C-terminal peptide in the protein structure acquisition, we studied in vitro refolding of the guanidinium chloride-denatured (K1K2)(2). Although they never reassociate with K2, in selected conditions the K1 fragments refold slowly to a dimeric state as shown by size exclusion chromatography data. This K1 dimer exhibits a fluorescence emission lambda max of 335 nm, a high degree of tyrosine exposure, strongly binds ANS but not MgADP, a CK substrate, and according to these structural characteristics, could be a dimeric molten globule species. We propose a folding model that takes into account the existence of a new transient intermediate state in the MM-CK refolding process. Besides two monomeric premolten and molten globule kinetic intermediates and the active final dimeric form, an inactive dimer, with partly compacted monomers, must ephemerally exist. Our results strongly suggest that the C-terminal end of the protein accelerates folding and plays a critical role for monomer final packing into a native-like conformation. The data also indicate that MM-CK catalytic efficiency is only acquired after dimerization.
UMR CNRS 5013, Biomembranes et Enzymes associés, Université Claude Bernard Lyon I, 43 boulevard du 11 Novembre 1918, 69622 Villeurbanne Cedex, France.
Hortense
H
Mazon
Olivier
O
Marcillat
Christian
C
Vial
Eric
E
Clottes
D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003402:Creatine Kinase / Q000737:chemistry*; D007700:Kinetics; D009132:Muscles / Q000201:enzymology*; D011489:Protein Denaturation; D017510:Protein Folding; D013050:Spectrometry, Fluorescence
D003402:Creatine Kinase