Johnson C M, Fersht A R
Cambridge Centre for Protein Engineering, England, U.K.
Biochemistry. 1995 May 23;34(20):6795-804. doi: 10.1021/bi00020a026.
The conventional procedure for analyzing urea denaturation curves assumes that the free energy of unfolding (delta GU-F) is linearly related to [urea] that is, delta GU-F = delta GH2O(U-F)--m[urea], where m is a constant, specific for each protein, and delta GH2O(U-F) is the free energy of unfolding in water. This relationship can be measured directly, however, over only a small concentration range of approximately +/- 0.8 M urea around the midpoint of the unfolding transition. A nagging discrepancy (1.6 kcal mol-1) between delta GH2O(U-F) at 298 K of barnase extrapolated from such an equation and the equivalent value obtained from thermal unfolding measurements has stimulated a re-evaluation of the equation. Differential scanning calorimetric measurements have been made of the thermal unfolding of barnase in the presence of concentrations of urea between 0 and 4.5 M, the midpoint of the unfolding transition at 298 K, to test the denaturation equation over a wide range of [urea]. Values for delta GU-F at 298 K (delta G298U-F) for each concentration of urea were extrapolated from the calorimetrically measured enthalpies and the denaturational heat capacity change (delta Cdp) measured for that concentration of urea. A plot of delta G298U-F against [urea] deviates systematically from linearity and fits better the equation: delta G298U-F = 10.5 +/- 0.08 - ((2.65 +/- 0.05) x [urea]) + ((0.08 +/- 0.01) x [urea]2) kcal mol-1. The curvature in the plot leads to apparent values of m that increase when measurements are made at lower concentrations of urea. This could account for increases in m at low values of pH or in destabilized mutants since the protein denatures at lower concentrations of urea. It has been shown previously that small curvature in the free energy of unfolding versus [urea] leads to negligible errors in measurements of delta delta GU-F, the change in free energy of unfolding on mutation, providing that the curvature is similar for all mutants. The calorimetrically measured enthalpies of unfolding are decreased in the presence of urea while delta Cdp is increased. Both of these observations are consistent with an overall exothermic interaction between urea and protein with a net increase on unfolding.
分析尿素变性曲线的传统方法假定去折叠自由能（ΔGU-F）与[尿素]呈线性关系，即ΔGU-F = ΔGH2O(U-F) – m[尿素]，其中m是一个常数，因蛋白质而异，而ΔGH2O(U-F)是在水中的去折叠自由能。然而，这种关系只能在去折叠转变中点周围约±0.8 M尿素的小浓度范围内直接测量。从该方程外推得到的298 K时巴纳酶的ΔGH2O(U-F)与通过热去折叠测量得到的等效值之间存在恼人的差异（1.6 kcal mol-1），这促使人们对该方程进行重新评估。已在0至4.5 M尿素浓度（298 K时去折叠转变的中点）存在的情况下对巴纳酶进行差示扫描量热测量，以在广泛的[尿素]范围内测试变性方程。从量热测量的焓和该尿素浓度下测量的变性热容量变化（ΔCdp）外推得到每种尿素浓度在298 K时的ΔGU-F值（ΔG298U-F）。ΔG298U-F对[尿素]的作图系统地偏离线性，并且更符合以下方程：ΔG298U-F = 10.5 ± 0.08 - ((2.65 ± 0.05) × [尿素]) + ((0.08 ± 0.01) × [尿素]2) kcal mol-1。该作图中的曲率导致m的表观值在较低尿素浓度下测量时增加。这可以解释在低pH值或不稳定突变体中m的增加，因为蛋白质在较低尿素浓度下去折叠。先前已表明，去折叠自由能对[尿素]的小曲率在测量ΔΔGU-F（突变时去折叠自由能的变化）时导致可忽略不计的误差，前提是所有突变体的曲率相似。在有尿素存在的情况下，量热测量的去折叠焓降低，而ΔCdp增加。这两个观察结果都与尿素和蛋白质之间的总体放热相互作用一致，且去折叠时净增加。
