The refolding of ubiquitin as site-directed mutagenesis and stopped-flow kinetic studies
Chung-Tien Lee1,2, Pei-Yeh Chen1, and Sunney I. Chan1.
1Institute of Chemistry, Academia Sinica, Taipei, Taiwan, R.O.C.
2Department of Chemistry, National Taiwan University, Taipei, Taiwan, R.O.C.
Ubiquitin, a small and compact α+β protein without any disulfide linkages, mediates intracellular protein degradation and many important biological functions. The formation of a β turn in the N-terminal β-hairpin has been implicated in the transition state of ubiquitin folding pathway. From earlier studies, we have shown that the Gly-10 residue is important in the stability of the hairpin. Deleting Gly-10 destabilizes the β-hairpin structure. On the contrary, mutating Thr-9 to Asp-9 has the opposite effect. Phe-4, a residue on the strand of the hairpin that participates as part of a small exterior hydrophobic cluster, also appears to contribute the stability of the hairpin. Here, we compare the effects of these residues in the folding of ubiquitin by examining the refolding/unfolding kinetics of the mutated proteins: desG10/F45W, T9D/F45W, and F4A/F45W. F45W mutation was introduced in order to provide a good fluorescence probe. Kinetic refolding and unfolding studies of these mutants were monitored by stopped-flow circular dichroism and stopped-flow fluorescence spectroscopy. From the results obtained, we propose that the turn residues are more important than the strand residue in the formation of the transition state.