Nicholson H, Söderlind E, Tronrud D E, Matthews B W
Institute of Molecular Biology, University of Oregon, Eugene 97403.
J Mol Biol. 1989 Nov 5;210(1):181-93. doi: 10.1016/0022-2836(89)90299-4.
Non-glycine residues in proteins are rarely observed to have "left-handed helical" conformations. For glycine, however, this conformation is common. To determine the contributions of left-handed helical residues to the stability of a protein, two such residues in phage T4 lysozyme, Asn55 and Lys124, were replaced with glycine. The mutant proteins fold normally and are fully active, showing that left-handed non-glycine residues, although rare, do not have an indispensable role in the folding of the protein or in its activity. The thermodynamic stability of the Lys124 to Gly variant is essentially identical with that of wild-type lysozyme. The Asn55 to Gly mutant protein is marginally less stable (0.5 kcal/mol). These results indicate that the conformational energy of a glycine and a non-glycine residue in the left-handed helical conformation are very similar. This is consistent with some theoretical energy distributions, but is inconsistent with others, which suggest that replacements of the sort described here might increase the stability of the protein by up to 5 kcal/mol. Crystallographic analysis of the mutant proteins shows that the backbone conformation of the Lys124 to Gly variant is essentially identical with that of the wild-type structure. In the case of the Asn55 to Gly replacement, however, the (phi, psi) values of residue 55 change by about 20 degrees. This suggests that the energy minimum for left-handed glycine residues is not the same as that for non-glycine residues. This is strongly indicated also by a survey of accurately determined protein crystal structures, which suggests that the energy minimum for left-handed glycine residues is near (phi = 90 degrees, psi = 0 degrees), whereas that for non-glycine residues is close to (phi = 60 degrees, psi = 30 degrees). This apparent energy minimum for glycine is not clearly predicted by any of the theoretical (phi, psi) energy contour maps.
蛋白质中的非甘氨酸残基很少呈现“左手螺旋”构象。然而，对于甘氨酸而言，这种构象很常见。为了确定左手螺旋残基对蛋白质稳定性的贡献，噬菌体T4溶菌酶中的两个此类残基，即天冬酰胺55和赖氨酸124，被替换为甘氨酸。突变蛋白正常折叠且具有完全活性，这表明左手非甘氨酸残基虽然罕见，但在蛋白质折叠或其活性方面并非具有不可或缺的作用。赖氨酸124突变为甘氨酸的变体的热力学稳定性与野生型溶菌酶基本相同。天冬酰胺55突变为甘氨酸的突变蛋白稳定性略低（0.5千卡/摩尔）。这些结果表明，甘氨酸和左手螺旋构象中的非甘氨酸残基的构象能非常相似。这与一些理论能量分布一致，但与其他一些理论不一致，其他理论表明此处所述的替换可能会使蛋白质的稳定性提高多达5千卡/摩尔。突变蛋白的晶体学分析表明，赖氨酸124突变为甘氨酸的变体的主链构象与野生型结构基本相同。然而，在天冬酰胺55被替换为甘氨酸的情况下，残基55的（φ，ψ）值变化约20度。这表明左手甘氨酸残基的能量最小值与非甘氨酸残基的不同。对精确测定的蛋白质晶体结构的调查也有力地表明了这一点，该调查表明左手甘氨酸残基的能量最小值接近（φ = 90度，ψ = 0度），而非甘氨酸残基的能量最小值接近（φ = 60度，ψ = 30度）。甘氨酸的这种明显能量最小值并未被任何理论（φ，ψ）能量等高线图明确预测。
