Zhukovsky E A, Mulkerrin M G, Presta L G
Department of Protein Engineering, Genentech, Inc., South San Francisco, California 94080.
Biochemistry. 1994 Aug 23;33(33):9856-64. doi: 10.1021/bi00199a006.
In this work we have investigated the contribution to protein stability of residues forming the boundaries of alpha-helices. At the N-terminus of helix 2 of human growth hormone there are two residues, Ser71 and Glu74, which form two reciprocal hydrogen bonds between the side chains and the backbone nitrogens of either residue (the N-capping box). In order to evaluate the stabilizing effect of each hydrogen bond, site-directed mutagenesis was employed. In addition, the effect of side-chain negative charge on helix stabilization, via charge dipole interaction, was assessed. Ultraviolet spectroscopy and near- and far-UV CD spectroscopy as well as guanidine hydrochloride protein denaturation were used as assays to monitor the conformational and free energy of stabilization changes induced by the point mutations. The results of these experiments can be summarized as follows: (a) receptor binding studies showed that the tertiary conformation of each mutant was similar to that of the native hormone, (b) far-UV CD spectroscopic analyses showed that the overall alpha-helical content was unchanged in the mutants, (c) UV absorption and CD spectroscopic analyses indicated small alterations in helical packing in those mutants in which the hydrogen bond between the side chain of Ser71 and backbone NH of Glu74 was disrupted, (d) the hydrogen bond involving the side chain of Ser71 contributes at least 1.0 kcal/mol to protein stabilization and has a 2-fold larger stabilizing effect than that of the hydrogen bond involving the Glu74 side chain, and (e) the putative charge-dipole interaction of Glu74 with the alpha-helix dipole does not contribute to the stabilization of the tertiary conformation of human growth hormone.
在这项研究中，我们调查了构成α-螺旋边界的残基对蛋白质稳定性的贡献。在人生长激素螺旋2的N端有两个残基，Ser71和Glu74，它们在两个残基的侧链与主链氮原子之间形成了两个相互的氢键（N-封端盒）。为了评估每个氢键的稳定作用，我们采用了定点诱变技术。此外，还评估了通过电荷-偶极相互作用，侧链负电荷对螺旋稳定性的影响。紫外光谱、近紫外和远紫外圆二色光谱以及盐酸胍蛋白变性实验被用作监测点突变引起的构象和稳定自由能变化的分析方法。这些实验结果可总结如下：（a）受体结合研究表明，每个突变体的三级构象与天然激素相似；（b）远紫外圆二色光谱分析表明，突变体中总的α-螺旋含量没有变化；（c）紫外吸收和圆二色光谱分析表明，在Ser71侧链与Glu74主链NH之间的氢键被破坏的那些突变体中，螺旋堆积有微小变化；（d）涉及Ser71侧链的氢键对蛋白质稳定性的贡献至少为1.0千卡/摩尔，其稳定作用比涉及Glu74侧链的氢键大2倍；（e）Glu74与α-螺旋偶极之间假定的电荷-偶极相互作用对人生长激素三级构象的稳定没有贡献。
