Spek E J, Bui A H, Lu M, Kallenbach N R
Department of Chemistry, New York University, New York 10003, USA.
Protein Sci. 1998 Nov;7(11):2431-7. doi: 10.1002/pro.5560071121.
We present a study of the role of salt bridges in stabilizing a simplified tertiary structural motif, the coiled-coil. Changes in GCN4 sequence have been engineered that introduce trial patterns of single and multiple salt bridges at solvent exposed sites. At the same sites, a set of alanine mutants was generated to provide a reference for thermodynamic analysis of the salt bridges. Introduction of three alanines stabilizes the dimer by 1.1 kcal/mol relative to the wild-type. An arrangement corresponding to a complex type of salt bridge involving three groups stabilizes the dimer by 1.7 kcal/ mol, an apparent elevation of the melting temperature relative to wild type of about 22 degrees C. While identifying local from nonlocal contributions to protein stability is difficult, stabilizing interactions can be identified by use of cycles. Introduction of alanines for side chains of lower helix propensity and complex salt bridges both stabilize the coiled-coil, so that combining the two should yield melting temperatures substantially higher than the starting species, approaching those of thermophilic sequences.
我们展示了一项关于盐桥在稳定一种简化的三级结构基序——卷曲螺旋中所起作用的研究。对GCN4序列进行了工程改造，在溶剂暴露位点引入了单个和多个盐桥的试验模式。在相同位点，生成了一组丙氨酸突变体，为盐桥的热力学分析提供参考。相对于野生型，引入三个丙氨酸可使二聚体稳定1.1千卡/摩尔。一种对应于涉及三个基团的复杂类型盐桥的排列使二聚体稳定1.7千卡/摩尔，相对于野生型，其熔解温度明显升高约22摄氏度。虽然很难区分对蛋白质稳定性的局部和非局部贡献，但可以通过使用循环来识别稳定相互作用。用螺旋倾向较低的侧链引入丙氨酸和复杂盐桥都能稳定卷曲螺旋，因此将两者结合应该会产生远高于起始物种的熔解温度，接近嗜热序列的熔解温度。
