dissimilarity in the folding of human cytosolic creatine kinase isoenzymes不同的人类胞质肌酸激酶同功酶的折叠.pdfVIP

Dissimilarity in the Folding of Human Cytosolic Creatine Kinase Isoenzymes 1,2 1 1,2 1,2 2,3 1 Yin Wang , Sha Wang , Yan-Song Gao , Zhe Chen , Hai-Meng Zhou , Yong-Bin Yan * 1 State Key Laboratory of Biomembrane and Membrane Biotechnology, School of Life Sciences, Tsinghua University, Beijing, China, 2 Protein Science Laboratory of the Ministry of Education, School of Life Sciences, Tsinghua University, Beijing, China, 3 Zhejiang Provincial Key Laboratory of Applied Enzymology, Yangtze Delta Region Institute of Tsinghua University, Zhejiang, China Abstract Creatine kinase (CK, EC ) 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