Clarke J, Henrick K, Fersht A R
Centre for Protein Engineering, MRC Centre, Cambridge, UK.
J Mol Biol. 1995 Oct 27;253(3):493-504. doi: 10.1006/jmbi.1995.0568.
In this series of papers, we examine the effects of introducing disulfide bonds on the properties, structure and thermodynamics of a small globular protein, barnase. Three mutants have been made, in each of which a single crosslink confers different properties. Two of the disulfide bonds, between residues 43 and 80 (43-80) and between residues 85 and 102 (85-102), stabilise the protein, relative to both wild-type and the corresponding (reduced) dithiol forms: 85-102 is more stable than predicted from the entropic destabilisation of the unfolded state; 43-80 is less stable than predicted. The third disulfide bond, between residues 70 and 92 (70-92) destabilises the protein relative to both wild-type and the corresponding dithiol form, implying significant disruption of the folded protein on formation of the disulfide bond. Crystal structures of the three mutant proteins have been solved. All three proteins have essentially the same fold as wild-type, but with left-handed disulfide bonds, which have dihedral geometries that have not been observed in naturally occurring disulfides. In the very stable mutant 85-102, there is no significant difference between the mutant and wild-type structures: these data do not explain the large stability of this protein. The disulfide bond at 43-80 induces small structural rearrangements close to the site of the disulfide bond, associated with some local disorder: the crosslink appears to decrease the stability of the native form of the protein. The destabilising disulfide bond at 70-92 induces considerable structural change, with displacement of a loop and consequent disruption of a stabilising salt-bridge. Our studies do not support the view that the conformation of the disulfide bond is crucial in determining the stability of the mutant proteins.
在这一系列论文中，我们研究了引入二硫键对一种小的球状蛋白——芽孢杆菌RNA酶（barnase）的性质、结构和热力学的影响。我们构建了三个突变体，每个突变体中的单个交联赋予了不同的性质。相对于野生型和相应的（还原的）二硫醇形式，其中两个二硫键，即残基43和80之间（43 - 80）以及残基85和102之间（85 - 102）的二硫键，使蛋白质稳定：85 - 102比根据未折叠状态的熵失稳预测的更稳定；43 - 80比预测的稳定性低。第三个二硫键，即残基70和92之间（70 - 92）的二硫键，相对于野生型和相应的二硫醇形式使蛋白质不稳定，这意味着在形成二硫键时折叠蛋白受到了显著破坏。已解析出这三种突变蛋白的晶体结构。所有这三种蛋白的折叠方式与野生型基本相同，但具有左手二硫键，其具有在天然存在的二硫键中未观察到的二面角几何结构。在非常稳定的突变体85 - 102中，突变体和野生型结构之间没有显著差异：这些数据无法解释该蛋白的高稳定性。43 - 80处的二硫键在靠近二硫键位点处诱导了小的结构重排，伴有一些局部无序：该交联似乎降低了蛋白质天然形式的稳定性。70 - 92处使蛋白不稳定的二硫键诱导了相当大的结构变化，一个环发生位移，从而破坏了一个稳定的盐桥。我们的研究不支持二硫键的构象在决定突变蛋白稳定性方面起关键作用的观点。
