Refolding of the hyperthermophilic protein Ssh10b involves a kinetic dimeric intermediate |
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Authors: | Meng Ge Yong-Jin Mao Xian-Ming Pan |
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Institution: | (1) The Key Laboratory of Bioinformatics, Ministry of Education, Department of Biological Sciences and Biotechnology, Tsinghua University, 100084 Beijing, China;(2) National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, 100101 Beijing, China;(3) Graduate University of Chinese Academy of Sciences, 100049 Beijing, China |
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Abstract: | The α/β-mixed dimeric protein Ssh10b from the hyperthermophile Sulfolobus shibatae is a member of the Sac10b family that is thought to be involved in chromosomal organization or DNA repair/recombination.
The equilibrium unfolding/refolding of Ssh10b induced by denaturants and heat was fully reversible, suggesting that Ssh10b
could serve as a good model for folding/unfolding studies of protein dimers. Here, we investigate the folding/unfolding kinetics
of Ssh10b in detail by stopped-flow circular dichroism (SF-CD) and using GdnHCl as denaturant. In unfolding reactions, the
native Ssh10b turned rapidly into fully unfolded monomers within the stopped-flow dead time with no detectable kinetic intermediate,
agreeing well with the results of equilibrium unfolding experiments. In refolding reactions, two unfolded monomers associate
in the burst phase to form a dimeric intermediate that undergoes a further, slower, first-order folding process to form the
native dimer. Our results demonstrate that the dimerization is essential for maintaining the native tertiary interactions
of the protein Ssh10b. In addition, folding mechanisms of Ssh10b and several other α/β-mixed or pure β-sheet proteins are
compared. |
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Keywords: | Protein dimer Folding kinetics Dimeric intermediate α /β -Mixed protein Stopped-flow circular dichroism |
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