Veltman O R, Vriend G, Hardy F, Mansfeld J, van den Burg B, Venema G, Eijsink V G
Department of Genetics, Biomolecular Sciences and Biotechnology Institute, University of Groningen, Haren, The Netherlands.
Eur J Biochem. 1997 Sep 1;248(2):433-40. doi: 10.1111/j.1432-1033.1997.00433.x.
Site-directed mutagenesis was used to assess the contribution of individual residues and a bound calcium in the 55-69 region of the thermolysin-like protease of Bacillus stearothermophilus (TLP-ste) to thermal stability. The importance of the 55-69 region was reflected by finding that almost all mutations had drastic effects on stability. These effects (both stabilizing and destabilizing) were obtained by mutations affecting main chain flexibility, as well as by mutations affecting the interaction between the 55-69 region and the rest of the protease molecule. The calcium-dependency of stability could be largely abolished by mutating one of its ligands (Asp57 or Asp59). In the case of the Asp57-->Ser mutation, the accompanying loss in stability was modest compared with the effects of other destabilizing mutations or the effects of (combinations of) stabilizing mutations. The detailed knowledge of the stability-determining region of TLP-ste permits effective rational design of stabilizing mutations, which, presumably, are also useful for related TLP such as thermolysin. This is demonstrated by the successful design of a stabilizing salt bridge involving residues 65 and 11.
定点诱变用于评估嗜热脂肪芽孢杆菌嗜热菌蛋白酶样蛋白酶（TLP-ste）55-69区域中单个残基和结合钙对热稳定性的贡献。几乎所有突变对稳定性都有显著影响，这反映了55-69区域的重要性。这些影响（包括稳定和不稳定）是通过影响主链柔韧性的突变以及影响55-69区域与蛋白酶分子其余部分之间相互作用的突变获得的。通过突变其一个配体（Asp57或Asp59），可以在很大程度上消除稳定性对钙的依赖性。在Asp57→Ser突变的情况下，与其他不稳定突变的影响或稳定突变（组合）的影响相比，伴随的稳定性损失较小。对TLP-ste稳定性决定区域的详细了解允许对稳定突变进行有效的合理设计，推测这对相关的TLP如嗜热菌蛋白酶也有用。涉及残基65和11的稳定盐桥的成功设计证明了这一点。
