Koh J T, Cornish V W, Schultz P G
Department of Chemistry, Howard Hughes Medical Institute, University of California, Berkeley, California 94720, USA.
Biochemistry. 1997 Sep 23;36(38):11314-22. doi: 10.1021/bi9707685.
The contribution of backbone hydrogen bonds in alpha-helices to the overall stability of a protein has been examined experimentally by replacing several backbone amide linkages in alpha-helix 39-50 of T4 lysozyme with ester linkages. T4 lysozyme variants wherein the backbone amide bonds between residues Ser38 and Leu39, Lys43 and Leu44, or Ala49 and Ile50 are replaced with ester bonds were generated by incorporating alpha-hydroxy acids at positions 39, 44, or 50, respectively, using unnatural amino acid mutagenesis. The stabilities of the proteins were determined from their thermal denaturation curves as monitored by circular dichroism. Comparison of the thermal stabilities of the amide- and ester-containing proteins shows that the ester substitution has a similar thermodynamic effect at all three positions. At the N- and C-terminal positions, where only one hydrogen-bonding interaction is perturbed, the ester substitution is destabilizing by 0.9 and 0.7 kcal/mol, respectively. Introduction of the ester linkage in the middle of the helix, which alters two hydrogen-bonding interactions, destabilizes the protein by 1.7 kcal/mol. The values obtained from these ester to amide mutations are compared to the values from similar mutations that have been made in other secondary structures and bimolecular complexes.
通过用酯键取代T4溶菌酶α-螺旋39 - 50中的几个主链酰胺键，实验研究了α-螺旋中主链氢键对蛋白质整体稳定性的贡献。利用非天然氨基酸诱变，分别在39、44或50位掺入α-羟基酸，生成了Ser38与Leu39、Lys43与Leu44或Ala49与Ile50之间的主链酰胺键被酯键取代的T4溶菌酶变体。通过圆二色性监测蛋白质的热变性曲线来确定其稳定性。含酰胺和含酯蛋白质热稳定性的比较表明，酯取代在所有三个位置都有相似的热力学效应。在N端和C端位置，仅一个氢键相互作用受到干扰，酯取代分别使稳定性降低0.9和0.7千卡/摩尔。在螺旋中间引入酯键会改变两个氢键相互作用，使蛋白质稳定性降低1.7千卡/摩尔。将这些酯到酰胺突变得到的值与在其他二级结构和双分子复合物中进行的类似突变的值进行比较。
