Gibbs Free Energy of Folding/Unfolding (ΔG)


Circular Dichroism (CD)


Mutations in the codon corresponding to positions 11 and 34 in the amino acid sequence of yeast ubiquitin were introduced as described. The incorporation of mutations was confirmed by sequencing all of the variants of the ubiquitin gene using the dideoxy chain termination sequencing method on an ABI PRISM 377 DNA sequencer. Overexpression of the ubiquitin variants was done in BL21(DE3) or JM109(DE3) strains of Escherichia coli. Proteins were purified to apparent homogeneity as described. The concentration of the
ubiquitin variants was measured spectrophotometrically using a molar extinction coefficient of epsilon280nm=1280. Correction for light-scattering was taken into account as described. Urea-induced unfolding of the ubiquitin variants was monitored by following the changes in ellipticity at 222 nm using a JASCO J-715 spectropolarimeter