Gibbs Free Energy of Folding/Unfolding (ΔG)
hypothesized that certain mutants might be identified that satisfy the condition Wa = fN,a, and if these backgrounds are generally noninteracting other than through stability effects, we can estimate fN,ab = Wab / Wb. The predicted fN,ab can then be used to estimate structural stability of single mutants (b)by DDGU = 2RT 3 ln(fU,ab / fN,ab)+ RT 3 ln(fU,a / fN,a). The large number of GB1 variants characterized in the liter-
ature provided a substantial reference to identify stability effects from the binding data. An automated analysis was performed that identified multiple background mutations (a) that generated DDGU values that correlate well with the values found in the literature. These backgrounds therefore satisfy the conditions stated above. This method is limited however to mutants (b) with sufficient fitness to produce a dynamic range in observed fitness (ab; those withWb < 0.24 correlated poorly). An average of the values generated from five reference backgrounds (Y3A, Y3C, L5N, L5S, and F30N) was highly correlated to 82 DDGU values published in the literature with a slope of 0.94 and a correlation coefficient (R) of 0.907.