Conformational Change in the C-Terminal Domain Is Responsible for the Initiation of Creatine Kinase Thermal Aggregation

¹ Tsinghua University

^* To whom correspondence should be addressed. E-mail: ybyan{at}tsinghua.edu.cn.

Submitted on May 9, 2005
Revised on June 6, 2005
Accepted on 29 June 2005

	Abstract

Protein conformational changes may be associated with particular properties such as its function, transportation, assembly, tendency to aggregation and potential cytotoxicity. In this research, the conformational change that is responsible for the fast destabilization and aggregation of rabbit muscle creatine kinase (RMCK, EC 2.7.3.2) induced by heat was studied by intrinsic fluorescence and infrared spectroscopy. A pretransitional change of the tryptophan microenvironments was found from the intrinsic fluorescence spectra. A further analysis of the infrared spectra using quantitative second derivative and two-dimensional correlation analysis indicated that the change of the -sheet structures in the C-terminal domain and the loops occurred prior to the formation of intermolecular cross -sheet structures and the unfolding of -helices. These results suggested that the pretransitional conformational changes in the active site and the C-terminal domain might result in the modification of the domain-domain interactions and the formation of an inactive dimeric form that is prone to aggregate. Our results highlighted the fact that some minor conformational changes, which were usually negligible or undetectable by normal methods, might play a crucial role in protein stability and aggregation. Our results also suggested that the change in domain-domain interactions, but not the dissociation of the dimer might play a crucial role in the thermal denaturation and aggregation of this dimeric two-domain protein.

Key Words: Fourier transform infrared spectroscopy, creatine kinase, domain-domain interactions, intrinsic fluorescence spectroscopy, pretransitional conformational change, protein aggregation

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