To provide a fluorescence probe for equilibrium and kinetic folding studies on ubiquitin, cassette mutagenesis in an Escherichia coli expression plasmid was used to replace the largely buried Phe 45 by a tryptophan. Under native conditions, the tryptophan fluorescence spectrum of this F45W mutant exhibits a blue-shifted emission maximum at 336 nm indicative of a largely solvent-shielded tryptophan environment. In contrast, the unfolded protein in 6 M guanidine hydrochloride (GuHCl) shows a 4-fold more intense emission band at 353 nm matching that of free tryptophan. The two-dimensional 1H NMR spectrum of F45W ubiquitin was assigned by comparison with published assignments of the wild type. The mutation results in only limited chemical shift changes for residues in the immediate vicinity of residue 45. The structural similarity of F45W with wild-type ubiquitin was confirmed by a preliminary analysis of the nuclear Overhauser spectrum. NMR and circular dichroism measurements of the reversible GuHCl-induced unfolding transition show that the F45W mutation lowers the stability of the folded ubiquitin structure by less than 0.4 kcal/mol. The biological activity of the mutant was found to be indistinguishable from that of wild-type in terms of its reaction with the ubiquitin activating enzyme E1 and an in vitro assay of ATP-dependent protein degradation. The kinetics of folding and unfolding of F45W ubiquitin was studied at two temperatures (8 and 25 degrees C) in a series of fluorescence-detected stopped-flow measurements over a wide range of GuHCl concentrations (0.5-6 M). The measurements at 25 degrees C are consistent with a two-state model with strongly denaturant-dependent folding and unfolding rates above about 2 M GuHCl. However, at lower denaturant concentrations, the rate of the major folding phase becomes GuHCl-independent, and up to 60% of the total fluorescence change occurs during the 2-ms dead time of the stopped-flow measurement. These observations provide clear evidence for the formation of an early folding intermediate during the first few milliseconds of refolding with a partially developed hydrophobic core involving Trp 45. The sigmoid denaturant dependence of the initial amplitude with an apparent midpoint of 1.3 M GuHCl suggests the presence of a discrete state that is destabilized at higher denaturant concentrations. In contrast, there is no evidence for an early intermediate in the folding kinetics at 8 degrees C. The destabilization of the intermediate at low temperature is consistent with a collapsed state stabilized primarily by hydrophobic interactions.
Submitter: Connie Wang
Submission Date: Aug. 9, 2017, 11:42 a.m.