Monomeric creatine kinase aggregation and sodium dodecyl sulfate-cyclodextrin assisted refolding.
The monomeric state of creatine kinase (CK) was stably captured at the equilibrium state by employing cysteine residue modifications in the presence of a denaturant, and at a partially folded state. The partially folded monomeric CK (PF-CK) was aggregated with kinetic order, which was mainly caused by the hydrophobic surface interactions between the CK subunits. The artificial chaperone, described as a SDS-cyclodextrin, was applied to prevent aggregation as well as to refold the PF-CK: SDS treatment onto the monomeric CK can significantly block aggregation and can be successfully refolded in the solutions containing cyclodextrins and DTT. Three types of cyclodextrins such as alpha-, beta-, and gamma-cyclodextrins were applied to strip SDS from the enzyme molecule, and each kinetic course was measured. The intrinsic fluorescence changes showed that reactivation occurred and this accompanied the conformational changes. The size exclusion chromatography detected the variously trapped monomeric CKs such as the thiol residue modified PF-CK, the SDS-binding PF-CK, the cyclodextrin treated PF-CK, and the DTT treated SDS-binding PF-CK. Our study demonstrated monomer CK aggregation for the first time; we also demonstrated the complex reassociation of CK during refolding with the aid of the SDS-cyclodextrin, and these pathways followed first-order kinetics.
Zou
He-Chang
HC
Department of Biological Science and Biotechnology, Tsinghua University, Beijing 100084, P. R. China.
Yu
Zhen-Hang
ZH
Wang
Ye-Jing
YJ
Yang
Jun-Mo
JM
Zhou
Hai-Meng
HM
Meng
Fan-Guo
FG
Park
Yong-Doo
YD
D000818:Animals; D003402:Creatine Kinase / Q000737:chemistry* / Q000378:metabolism*; D003505:Cyclodextrins / Q000494:pharmacology*; D003545:Cysteine; D004789:Enzyme Activation; D007700:Kinetics; D018482:Muscle, Skeletal / Q000201:enzymology; D017510:Protein Folding; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D011817:Rabbits; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology*
D003505:Cyclodextrins; D021122:Protein Subunits; D012967:Sodium Dodecyl Sulfate; D003402:Creatine Kinase; D003545:Cysteine