title: Dissimilarity in the folding of human cytosolic creatine kinase isoenzymes.
abstract: Creatine kinase (CK, EC 2.7.3.2) plays a key role in the energy homeostasis of excitable cells. The cytosolic human CK isoenzymes exist as homodimers (HMCK and HBCK) or a heterodimer (MBCK) formed by the muscle CK subunit (M) and/or brain CK subunit (B) with highly conserved three-dimensional structures composed of a small N-terminal domain (NTD) and a large C-terminal domain (CTD). The isoforms of CK provide a novel system to investigate the sequence/structural determinants of multimeric/multidomain protein folding. In this research, the role of NTD and CTD as well as the domain interactions in CK folding was investigated by comparing the equilibrium and kinetic folding parameters of HMCK, HBCK, MBCK and two domain-swapped chimeric forms (BnMc and MnBc). Spectroscopic results indicated that the five proteins had distinct structural features depending on the domain organizations. MBCK BnMc had the smallest CD signals and the lowest stability against guanidine chloride-induced denaturation. During the biphasic kinetic refolding, three proteins (HMCK, BnMc and MnBc), which contained either the NTD or CTD of the M subunit and similar microenvironments of the Trp fluorophores, refolded about 10-fold faster than HBCK for both the fast and slow phase. The fast folding of these three proteins led to an accumulation of the aggregation-prone intermediate and slowed down the reactivation rate thereby during the kinetic refolding. Our results suggested that the intra- and inter-subunit domain interactions modified the behavior of kinetic refolding. The alternation of domain interactions based on isoenzymes also provides a valuable strategy to improve the properties of multidomain enzymes in biotechnology.
authors.affiliation: State Key Laboratory of Biomembrane and Membrane Biotechnology, School of Life Sciences, Tsinghua University, Beijing, China.
authors.forename: Yin
authors.initials: Y
authors.lastname: Wang
authors.forename: Sha
authors.initials: S
authors.lastname: Wang
authors.forename: Yan-Song
authors.initials: YS
authors.lastname: Gao
authors.forename: Zhe
authors.initials: Z
authors.lastname: Chen
authors.forename: Hai-Meng
authors.initials: HM
authors.lastname: Zhou
authors.forename: Yong-Bin
authors.initials: YB
authors.lastname: Yan
mesh_terms: D003402:Creatine Kinase / Q000737:chemistry* / Q000378:metabolism*; D003600:Cytosol / Q000201:enzymology*; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D017510:Protein Folding
chemical_list: D007527:Isoenzymes; D003402:Creatine Kinase