Zhang X J, Baase W A, Shoichet B K, Wilson K P, Matthews B W
Howard Hughes Medical Institute, University of Oregon, Eugene 97403, USA.
Protein Eng. 1995 Oct;8(10):1017-22. doi: 10.1093/protein/8.10.1017.
A number of mutations have been shown previously to stabilize T4 lysozyme. By combining up to seven such mutations in the same protein, the melting temperature was incrementally increased by up to 8.3 degrees C at pH 5.4 (delta delta G = 3.6 kcal/mol). This shows that it is possible to engineer a protein of enhanced thermostability by combining a series of rationally designed point mutations. It is also shown that this stabilization is achieved with only minor, localized changes in the structure of the protein. This is consistent with the observation that the change in stability of each of the multiple mutants is, in each case, additive, i.e. equal to the sum of the stability changes associated with the constituent single mutants. One of the seven substitutions, Asn116-->Asp, changes a residue that participates in substrate binding; not surprisingly, it causes a significant loss in activity. Ignoring this mutation, there is a gradual reduction in activity as successively more mutations are combined.
此前已证明，一些突变可使T4溶菌酶稳定。通过在同一蛋白质中组合多达七个此类突变，在pH 5.4时，解链温度逐渐升高，最高可达8.3摄氏度（ΔΔG = 3.6千卡/摩尔）。这表明，通过组合一系列合理设计的点突变，可以构建出热稳定性增强的蛋白质。研究还表明，这种稳定性的提高仅伴随着蛋白质结构的微小局部变化。这与以下观察结果一致：在每种情况下，多个突变体中每个突变体稳定性的变化都是累加的，即等于与组成单个突变体相关的稳定性变化之和。七个取代之一，Asn116→Asp，改变了一个参与底物结合的残基；不出所料，它导致活性显著丧失。忽略此突变，随着组合的突变数量相继增加，活性会逐渐降低。
