Johnson C M, Oliveberg M, Clarke J, Fersht A R
Cambridge Centre for Protein Engineering, UK.
J Mol Biol. 1997 Apr 25;268(1):198-208. doi: 10.1006/jmbi.1997.0928.
We have measured the effects of disulfide crosslinks on the thermodynamics of denaturation of three mutants of barnase that contain cystine and the corresponding single and double cysteine mutants. At first sight, the data are consistent with the hypothesis that disulfide crosslinks stabilise proteins through entropic destabilisation of the denatured state, but the decreases in the entropy of denaturation are larger than predicted and are accompanied by decreases in the enthalpy of denaturation. These effects are not a unique feature of the disulfide crosslink and are observed in a range of non-crosslinked mutants of barnase as part of a general enthalpy-entropy compensation phenomenon. Similarly, effects on the heat capacity change for denaturation (delta C(p)d), determined from the slope of the enthalpy of denaturation versus temperature, are not confined to mutants with disulfide crosslinks. The value of delta C(p)d is lower in four stabilised mutants than in wild-type barnase, irrespective of the presence of a disulfide crosslink, while the delta C(p)d remains unchanged in a destabilised mutant containing a disulfide. The variation in delta C(p)d may result from an inherent temperature-dependence of delta C(p)d, since it is measured for each mutant over a different temperature range. The thermodynamics of denaturation of the disulfide mutant with a crosslink between positions 70 and 92 change anomalously with pH but in a similar way to that of the D93N mutant of barnase, which lacks the D93-R69 salt-bridge present in the wild-type. This finding confirms initial observations in the X-ray structure of this disulfide mutant that the salt-bridge has been disrupted by the introduced crosslink.
我们已经测量了二硫键交联对三种含有胱氨酸的巴纳酶突变体以及相应的单半胱氨酸和双半胱氨酸突变体变性热力学的影响。乍一看，数据与二硫键交联通过使变性状态的熵不稳定来稳定蛋白质的假设一致，但变性熵的降低比预期的要大，并且伴随着变性焓的降低。这些效应并非二硫键交联所特有，在一系列巴纳酶非交联突变体中也观察到了，这是一般焓 - 熵补偿现象的一部分。同样，由变性焓对温度的斜率确定的变性热容量变化（ΔC(p)d）的影响并不局限于具有二硫键交联的突变体。无论是否存在二硫键交联，四个稳定突变体中的ΔC(p)d值都低于野生型巴纳酶，而在一个含有二硫键的不稳定突变体中，ΔC(p)d保持不变。ΔC(p)d的变化可能源于ΔC(p)d固有的温度依赖性，因为它是在不同温度范围内对每个突变体进行测量的。在位置70和92之间有交联的二硫键突变体的变性热力学随pH值异常变化，但与巴纳酶的D93N突变体类似，该突变体缺乏野生型中存在的D93 - R69盐桥。这一发现证实了对该二硫键突变体X射线结构的初步观察结果，即引入的交联破坏了盐桥。
