Veltman O R, Vriend G, Middelhoven P J, van den Burg B, Venema G, Eijsink V G
Department of Genetics, University of Groningen, Haren, The Netherlands.
Protein Eng. 1996 Dec;9(12):1181-9. doi: 10.1093/protein/9.12.1181.
The thermolysin-like protease (TLP) produced by Bacillus stearothermophilus CU21 (TLP-ste) differs at 43 positions from the more thermally stable thermolysin (containing 316 residues in total). Of these differences, 26 were analysed by studying the effect of replacing residues in TLP-ste by the corresponding residues in thermolysin. Several stabilizing mutations were identified but, remarkably, considerable destabilizing mutational effects were also found. A Tyr-rich three residue insertion in TLP-ste (the only deletional/insertional difference between the two enzymes) appeared to make an important contribution to the stability of the enzyme. Mutations with large effects on stability were all localized in the beta-pleated N-terminal domain of TLP-ste, confirming observations that this domain has a lower intrinsic stability than the largely alpha-helical C-terminal domain. Rigidifying mutations such as Gly58-->Ala and Ala69-->Pro were among the most stabilizing ones. Apart from this observation, the analyses did not reveal general rules for stabilizing proteins. Instead, the results highlight the importance of context in evaluating the stability effects of mutations.
嗜热脂肪芽孢杆菌CU21产生的嗜热菌蛋白酶样蛋白酶（TLP-ste）与热稳定性更高的嗜热菌蛋白酶（总共含有316个残基）在43个位置上存在差异。在这些差异中，通过研究用嗜热菌蛋白酶中的相应残基替换TLP-ste中的残基的效果，对其中26个差异进行了分析。确定了几个稳定化突变，但值得注意的是，也发现了相当大的去稳定化突变效应。TLP-ste中富含酪氨酸的三个残基插入（这是两种酶之间唯一的缺失/插入差异）似乎对该酶的稳定性有重要贡献。对稳定性有较大影响的突变都位于TLP-ste的β折叠N端结构域，这证实了该结构域的固有稳定性低于主要为α螺旋的C端结构域的观察结果。诸如Gly58→Ala和Ala69→Pro等刚性化突变是最具稳定化作用的突变之一。除了这一观察结果外，分析并未揭示稳定蛋白质的一般规律。相反，结果突出了背景在评估突变的稳定性效应方面的重要性。
