Gassner Nadine C, Baase Walter A, Mooers Blaine H M, Busam Robert D, Weaver Larry H, Lindstrom Joel D, Quillin Michael L, Matthews Brian W
Institute of Molecular Biology, Howard Hughes Medical Institute, 1229 University of Oregon, Eugene, OR 97403-1229, USA.
Biophys Chem. 2003;100(1-3):325-40. doi: 10.1016/s0301-4622(02)00290-9.
In order to further explore the tolerance of proteins to amino acid substitutions within the interior, a series of core residues was replaced by methionine within the C-terminal domain of T4 lysozyme. By replacing leucine, isoleucine, valine and phenylalanine residues a total of 10 methionines could be introduced, which corresponds to a third of the residues that are buried in this domain. As more methionines are incorporated the protein gradually loses stability. This is attributed in part to a reduction in hydrophobic stabilization, in part to the increased entropic cost of localizing the long, flexible methionine sidechains, and in part to steric clashes. The changes in structure of the mutants relative to the wildtype protein are modest but tend to increase in an additive fashion as more methionines are included. In the most extreme case, namely the 10-methionine mutant, much of the C-terminal domain remains quite similar to wildtype (root-mean-square backbone shifts of 0.56 A), while the F and G helices undergo rotations of approximately 20 degrees and center-of-mass shifts of approximately 1.4 A. For up to six methionine substitutions the changes in stability are additive. Beyond this point, however, the multiple mutants are somewhat more stable than suggested from the sum of their constituents, especially for those including the replacement Val111-->Met. This is interpreted in terms of the larger structural changes associated with this substitution. The substituted sidechains in the mutant structures have somewhat higher crystallographic thermal factors than their counterparts in WT*. Nevertheless, the interiors of the mutant proteins retain a well-defined structure with little suggestion of molten-globule characteristics. Lysozymes in which selenomethionine has been incorporated rather than methionine tend to have increased stability. At the same time they also fold faster. This provides further evidence that, at the rate-limiting step in folding, the structure of the C-terminal domain of T4 lysozyme is similar to that of the fully folded protein.
为了进一步探究蛋白质对内部氨基酸替换的耐受性，在T4溶菌酶的C端结构域内，一系列核心残基被甲硫氨酸取代。通过替换亮氨酸、异亮氨酸、缬氨酸和苯丙氨酸残基，总共可以引入10个甲硫氨酸，这相当于该结构域中埋藏残基的三分之一。随着更多甲硫氨酸的掺入，蛋白质逐渐失去稳定性。这部分归因于疏水稳定性的降低，部分归因于定位长而灵活的甲硫氨酸侧链的熵成本增加，部分归因于空间冲突。相对于野生型蛋白质，突变体的结构变化不大，但随着更多甲硫氨酸的加入，这些变化往往以累加的方式增加。在最极端的情况下，即10-甲硫氨酸突变体，大部分C端结构域仍与野生型非常相似（均方根主链位移为0.56 Å），而F和G螺旋经历了约20度的旋转和约1.4 Å的质心位移。对于多达六个甲硫氨酸替换，稳定性变化是累加的。然而，超过这一点后，多个突变体比其组成部分之和所暗示的要稍微稳定一些，特别是对于那些包括Val111→Met替换的突变体。这可以根据与该替换相关的更大结构变化来解释。突变体结构中的取代侧链比野生型中的对应侧链具有略高的晶体学热因子。尽管如此，突变体蛋白质的内部保留了明确的结构，几乎没有熔融球状体特征的迹象。掺入硒代甲硫氨酸而非甲硫氨酸的溶菌酶往往具有更高的稳定性。同时它们也折叠得更快。这提供了进一步的证据，即在折叠的限速步骤中，T4溶菌酶C端结构域的结构与完全折叠的蛋白质相似。
