title: Refolding of the hyperthermophilic protein Ssh10b involves a kinetic dimeric intermediate.
abstract: The alpha/beta-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 alpha/beta-mixed or pure beta-sheet proteins are compared.
authors.affiliation: The Key Laboratory of Bioinformatics, Ministry of Education, Department of Biological Sciences and Biotechnology, Tsinghua University, 100084 Beijing, China.
authors.forename: Meng
authors.initials: M
authors.lastname: Ge
authors.forename: Yong-Jin
authors.initials: YJ
authors.lastname: Mao
authors.forename: Xian-Ming
authors.initials: XM
authors.lastname: Pan
mesh_terms: D019843:Archaeal Proteins / Q000737:chemistry*; D002942:Circular Dichroism; D019281:Dimerization; D019791:Guanidine; D007700:Kinetics; D011489:Protein Denaturation; D017510:Protein Folding; D016944:Sulfolobus / Q000737:chemistry*; D014461:Ultracentrifugation
chemical_list: D019843:Archaeal Proteins; D019791:Guanidine