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Effect of Ficoll 70 on Thermal Stability and Structure of Creatine Kinase

Yejing Wang1, Huawei He2, and Sen Li3*

1School of Life Sciences, Lanzhou University, Lanzhou 730000, Gansu, P. R. China

2Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing 100084, P. R. China

3Department of Biochemistry and Molecular Biology, Beijing Normal University, Beijing Key Laboratory, Beijing 100875, P. R. China; fax: 86-10-5880-7720; E-mail: lisen@bnu.edu.cn

* To whom correspondence should be addressed.

Received January 27, 2010; Revision received February 24, 2010
The effect of Ficoll 70 on the thermal stability and structure of creatine kinase (CK) was studied using far-UV CD spectra and intrinsic fluorescence spectra. The thermal transition curves monitored by CD spectra were fitted to a two-state model using a modified form of the van’t Hoff equation to obtain the transition temperature (Tm) and enthalpy change (ΔHu) of thermally induced denaturation of CK in the absence and presence of Ficoll 70. An increase in Tm with constant ΔHu was observed with increasing Ficoll 70 concentration, suggesting that Ficoll 70 enhances the thermal stability of CK. Fluorescence spectral measurements confirmed this protective effect of Ficoll 70 on CK structure. In addition, we observed a crowding-induced compaction effect on the structure of both native state and thermally denatured state of CK in the presence of Ficoll 70, which is more obvious on the structure of the denatured ensemble compared to that of the native ensemble. Our observations qualitatively accord with the predictions of previously proposed crowding theory for the effect of intermolecular excluded volume on protein stability and structure. These findings imply that the effects of macromolecular crowding are essential to our understanding of protein folding and unfolding occurring in vivo.
KEY WORDS: macromolecular crowding, thermal stability, Ficoll 70, creatine kinase

DOI: 10.1134/S0006297910050160