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.