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Journal of Biochemistry Advance Access originally published online on April 3, 2009
Journal of Biochemistry 2009 146(2):193-200; doi:10.1093/jb/mvp055
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© The Authors 2009. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved

Effect of Metal Ion on the Structural Stability of Tumour Suppressor Protein p53 DNA-Binding Domain

Yonglai Xue, Shuai Wang and Xizeng Feng*

College of Life Science, Nankai University at Tianjin, P. R. China 300071

*To whom correspondence should be addressed. Tel: +86-22-2350-7022, Fax: +86-22-2350-7022, E-mail: xzfeng{at}nankai.edu.cn

Received March 9, 2009; Accepted March 24, 2009


  Abstract

The tumour suppressor protein p53 is a sequence-specific transcription factor that coordinates one molecule of zinc in the core domain. In our recent study, magnesium can also bind to the p53DBD and enhance its DNA-binding activity. In this study, a systematic analysis of the conformation and stability changes induced by these two metal ions was reported. The spectra of protein intrinsic fluorescence were used to measure the equilibrium unfolding of the p53DBD protein. The stability against chemical denaturation increased in the order apo < Mg2+ < Zn2+. The thermal stability monitored by DSC scans showed that the binding of metal ions to p53DBD increased the thermal stability of the protein. To explore additional information of structural changes after the binding of metal ions, we used the fluorescent probes to evaluate the hydrophobic surface exposure. The results established that metal ions binding increased hydrophobic exposure on the surface of p53DBD. Analysis of acrylamide quenching experiments revealed that the binding of metal ions to p53DBD induced a structural modification of the protein and this change provided significant protection against acrylamide quenching. Overall, the present results indicated that p53DBD underwent a conformational change upon the binding of metal ions, which was characterized by an increased stability of the protein.

Key Words: equilibrium unfolding, denature, fluorescence, conformational change, stability

Abbreviations: ANS, 1-anilino-8-naphthalene sulphonate; DBD, DNA-binding domain; DSC, differential scanning calorimetry; DTT, dithiothreitol; GdmHCl, guanidinium hydrochloride; SDS, sodium dodecyl sulphate


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