Evidence for kinetic intermediate states during the refolding of GdnHCl-denatured MM-creatine kinase. Characterization of a trapped monomeric species.
The kinetics of refolding of guanidinium chloride-denatured rabbit MM-creatine kinase was investigated. Recovery of enzymatic activity is biphasic, depending on the temperature but not on the protein or DTT concentration. Only 45% of the original, active dimeric form is recovered even after several hours of refolding. The reactivation yield is limited by the accumulation of a highly stable but nonproductive monomeric species. The ratio of "correct" to "incorrect" forms depends on the duration of exposure to the denaturant, which may be consistent with the existence of a heterogeneous population of unfolded states with regard to proline isomerization. The first fast reaction observed during renaturation results in the appearance of collapsed monomeric states, displaying features of a pre-molten globule state. These burst species are rapidly transformed into more structured monomers resembling a molten globule state possessing a partially folded C-terminal domain. A proportion of these latter transient intermediates (45%) associates into an active dimer, while the remainder (55%) is trapped by reshuffling in a monomeric dead-end product. Our results strongly indicate that (i) the dimeric state is a prerequisite for the expression of catalytic activity, (ii) the kinetic intermediates of refolding are very similar to those observed during equilibrium unfolding, and (iii) refolding of creatine kinase in these conditions is limited by the accumulation of inactive misfolded nondimerizable monomer.
Leydier
C
C
Biomembranes et Enzymes Associés, UPRESA 5013 Centre National de la Recherche Scientifique, Université Lyon I, Villeurbanne, France.
Clottes
E
E
Couthon
F
F
Marcillat
O
O
Ebel
C
C
Vial
C
C
D000817:Anilino Naphthalenesulfonates / Q000378:metabolism; D000818:Animals; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002850:Chromatography, Gel; D003402:Creatine Kinase / Q000737:chemistry* / Q000378:metabolism*; D003545:Cysteine / Q000737:chemistry; D004789:Enzyme Activation / Q000187:drug effects; D019791:Guanidine / Q000494:pharmacology*; D007527:Isoenzymes; D007700:Kinetics; D008956:Models, Chemical; D011485:Protein Binding / Q000187:drug effects; D011489:Protein Denaturation / Q000187:drug effects; D017510:Protein Folding; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D017186:Titrimetry; D014364:Tryptophan / Q000737:chemistry; D014443:Tyrosine / Q000737:chemistry
D000817:Anilino Naphthalenesulfonates; D007527:Isoenzymes; D014443:Tyrosine; C027132:1-anilino-8-naphthalenesulfonate; D014364:Tryptophan; D003402:Creatine Kinase; D019791:Guanidine; D003545:Cysteine