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Updated: Jun 28, 2026
13:52
Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae
Published on: July 9, 2013
Refolding of the hyperthermophilic protein Ssh10b involves a kinetic dimeric intermediate
Meng Ge1, Yong-Jin Mao, Xian-Ming Pan
1The Key Laboratory of Bioinformatics, Ministry of Education, Department of Biological Sciences and Biotechnology, Tsinghua University, 100084 Beijing, China.
Extremophiles : Life Under Extreme Conditions
|November 13, 2008
Summary
The protein Ssh10b dimer unfolds rapidly into monomers. Refolding involves dimerization followed by a slower folding step, highlighting dimerization
Area of Science:
- Protein folding and biophysics
- Biochemistry and molecular biology
- Structural biology
Background:
- Ssh10b is an alpha/beta-mixed dimeric protein from Sulfolobus shibatae, belonging to the Sac10b family.
- This protein family is implicated in chromosomal organization and DNA repair/recombination processes.
- Previous studies indicated Ssh10b's reversible unfolding/refolding, suggesting its utility in protein dimer folding studies.
Purpose of the Study:
- To kinetically investigate the folding and unfolding mechanisms of the Ssh10b dimer.
- To elucidate the role of dimerization in the protein's structural stability and folding pathway.
- To compare the folding kinetics of Ssh10b with other related protein structures.
Main Methods:
- Stopped-flow circular dichroism (SF-CD) spectroscopy was employed.
- Guanidinium hydrochloride (GdnHCl) was used as the denaturant to induce unfolding.
- Kinetic analysis of unfolding and refolding reactions was performed.
Main Results:
- Unfolding of native Ssh10b resulted in rapid conversion to fully unfolded monomers, with no observable kinetic intermediates.
- Refolding initiated with a burst-phase dimerization of unfolded monomers.
- A subsequent, slower, first-order process led to the formation of the native Ssh10b dimer.
Conclusions:
- Dimerization is a critical step for establishing and maintaining the native tertiary structure of Ssh10b.
- The folding pathway of Ssh10b involves distinct phases of monomer unfolding and dimer formation.
- Kinetic insights into Ssh10b folding contribute to understanding protein dimer dynamics and stability.
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