Role of the linker between the N- and C-terminal domains in the stability and folding of rabbit muscle creatine kinase.
Domain-domain interactions may be very important to the structure and functions of many multidomain proteins. However, little is known about the role of the linker in the folding, stability and function of multidomain proteins. In this research, muscle creatine kinase (CK), a dimeric two-domain protein, was used as a model protein to investigate the role of the linker in CK activity, stability and folding by mutational analysis. Two of the three mutations, L115D and L121D, resulted in a gradual decrease in CK activity and secondary structures, but did not affect CK inactivation induced by heat or guanidine hydrochloride (GdnHCl). The mutations also caused much more serious aggregation during heat- and GdnHCl-induced denaturation and refolding from the GdnHCl-denatured state. More importantly, none of the three mutants could successfully recover their activities by dilution-initiated refolding, and the rate constant of CK refolding was gradually decreased by the mutations. These results suggested that mutations of the hydrophobic residues in the linker might affect the correct positioning of the domains and thus disrupt the efficient recognition and interactions between the two domains. The results herein indicated that in addition to its role in the in vivo functions, the linker also played a crucial role in the stability and folding of CK.
He
Hua-Wei
HW
State Key Laboratory of Biomembrane and Membrane Biotechnology, Protein Science Laboratory of the Ministiry of Education, Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing 100084, China.
Feng
Shan
S
Pang
Min
M
Zhou
Hai-Meng
HM
Yan
Yong-Bin
YB
D000595:Amino Acid Sequence; D000818:Animals; D052283:Creatine Kinase, MM Form / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004795:Enzyme Stability; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000502:physiology; D011489:Protein Denaturation / Q000235:genetics; D017510:Protein Folding; D017434:Protein Structure, Tertiary / Q000502:physiology; D011817:Rabbits; D013696:Temperature
D052283:Creatine Kinase, MM Form