Ramachandran S, Udgaonkar J B
National Centre for Biological Sciences, TIFR Centre, Bangalore, India.
Biochemistry. 1996 Jul 2;35(26):8776-85. doi: 10.1021/bi9600759.
The internal packing of residues in the small monomeric protein barstar was severely perturbed by chemical modification of the two buried cysteine residues with the thiol reagent 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) after prior unfolding of the protein using guanidine hydrochloride (GdnHCl). The modification produces mixed disulfides between 5-thio(2-nitrobenzoic acid) and the two Cys residues. To understand the effects of the modification of the individual cysteine residues, Cys40 and Cys82, the modification was also carried out on the two single Cys --> Ala mutant forms of barstar, C40A and C82A, whose structures, activities, and stabilities were first shown to be similar to those of wt barstar. Equilibrium GdnHCl-induced denaturation studies on wt barstar show that the modification causes the midpoint of the denaturation curve to increase by 0.6 M and the stability to increase by 1.3 kcal mol-1. Both C40A and C82A also denature at higher concentrations of GdnHCl after modification. Modification of Cys40 has approximately the same stabilizing contribution as does modification of Cys82. The structures of the modified and unmodified proteins have been compared using circular dichroism (CD) spectroscopy, UV difference absorption spectroscopy, and fluorescence spectroscopy. It is shown that the 5-thio(2-nitrobenzoic acid) groups introduced by reaction with DTNB are buried in hydrophobic environments in the modified C40A and C82A mutant proteins, as well as in modified wt barstar. The far-UV CD spectra of the modified and unmodified proteins are similar, but the mean residue ellipticity at 220 nm of wt barstar is reduced by 30% upon modification. Such a decrease is not seen for either C40A or C82A. The barnase-inhibiting activities of the three modified proteins are shown to be similar to those of the corresponding unmodified proteins. Thus, the severe perturbations of the internal packing, which result in a significant increase in stability, do not appear to affect the overall fold of barstar.
在使用盐酸胍（GdnHCl）使小的单体蛋白芽孢杆菌RNA酶抑制剂（barstar）预先展开后，用硫醇试剂5,5'-二硫代双（2-硝基苯甲酸）（DTNB）对两个埋藏的半胱氨酸残基进行化学修饰，严重扰乱了barstar内部残基的堆积。该修饰在5-硫代（2-硝基苯甲酸）与两个半胱氨酸残基之间产生了混合二硫键。为了了解单个半胱氨酸残基Cys40和Cys82修饰的影响，还对barstar的两种单个半胱氨酸突变为丙氨酸的突变体形式C40A和C82A进行了修饰，其结构、活性和稳定性首先被证明与野生型barstar相似。对野生型barstar进行的平衡GdnHCl诱导变性研究表明，该修饰导致变性曲线的中点增加0.6 M，稳定性增加1.3 kcal mol-1。修饰后，C40A和C82A在更高浓度的GdnHCl下也会变性。Cys40的修饰与Cys82的修饰具有大致相同的稳定作用。使用圆二色性（CD）光谱、紫外差吸收光谱和荧光光谱对修饰和未修饰蛋白质的结构进行了比较。结果表明，与DTNB反应引入的5-硫代（2-硝基苯甲酸）基团在修饰的C40A和C82A突变体蛋白以及修饰的野生型barstar中都埋藏在疏水环境中。修饰和未修饰蛋白质的远紫外CD光谱相似，但野生型barstar在220 nm处的平均残基椭圆率在修饰后降低了30%。C40A和C82A均未出现这种降低。三种修饰蛋白对芽孢杆菌RNA酶（barnase）的抑制活性与相应未修饰蛋白的抑制活性相似。因此，内部堆积的严重扰动导致稳定性显著增加，但似乎并未影响barstar的整体折叠。
