Lipscomb L A, Gassner N C, Snow S D, Eldridge A M, Baase W A, Drew D L, Matthews B W
Institute of Molecular Biology, Howard Hughes Medical Institute and Department of Chemistry, University of Oregon, Eugene 97403, USA.
Protein Sci. 1998 Mar;7(3):765-73. doi: 10.1002/pro.5560070326.
The substitution of methionines with leucines within the interior of a protein is expected to increase stability both because of a more favorable solvent transfer term as well as the reduced entropic cost of holding a leucine side chain in a defined position. Together, these two terms are expected to contribute about 1.4 kcal/mol to protein stability for each Met --> Leu substitution when fully buried. At the same time, this expected beneficial effect may be offset by steric factors due to differences in the shape of leucine and methionine. To investigate the interplay between these factors, all methionines in T4 lysozyme except at the amino-terminus were individually replaced with leucine. Of these mutants, M106L and M120L have stabilities 0.5 kcal/mol higher than wild-type T4 lysozyme, while M6L is significantly destabilized (-2.8 kcal/mol). M102L, described previously, is also destabilized (-0.9 kcal/mol). Based on this limited sample it appears that methionine-to-leucine substitutions can increase protein stability but only in a situation where the methionine side chain is fully or partially buried, yet allows the introduction of the leucine without concomitant steric interference. The variants, together with methionine-to-lysine substitutions at the same sites, follow the general pattern that substitutions at rigid, internal sites tend to be most destabilizing, whereas replacements at more solvent-exposed sites are better tolerated.
在蛋白质内部用亮氨酸取代甲硫氨酸有望提高稳定性，这既是因为溶剂转移项更有利，也是因为将亮氨酸侧链固定在特定位置时熵成本降低。综合起来，当完全掩埋时，每一次甲硫氨酸到亮氨酸的取代，这两个因素预计会为蛋白质稳定性贡献约1.4千卡/摩尔。同时，由于亮氨酸和甲硫氨酸形状的差异，这种预期的有益效果可能会被空间因素抵消。为了研究这些因素之间的相互作用，除了氨基末端外，T4溶菌酶中的所有甲硫氨酸都被逐个替换为亮氨酸。在这些突变体中，M106L和M120L的稳定性比野生型T4溶菌酶高0.5千卡/摩尔，而M6L则显著失稳（-2.8千卡/摩尔）。之前描述过的M102L也失稳了（-0.9千卡/摩尔）。基于这个有限的样本，似乎甲硫氨酸到亮氨酸的取代可以提高蛋白质稳定性，但只有在甲硫氨酸侧链完全或部分被掩埋的情况下，同时引入亮氨酸时不会伴随空间干扰。这些变体，以及在相同位点的甲硫氨酸到赖氨酸的取代，遵循一般模式，即在刚性的内部位点进行取代往往最容易导致失稳，而在更多暴露于溶剂的位点进行取代则更能被容忍。
