Pradeep Lovy, Kurinov Igor, Ealick Steven E, Scheraga Harold A
Baker Laboratory of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 14853, USA.
Structure. 2007 Oct;15(10):1178-89. doi: 10.1016/j.str.2007.08.003.
A previously introduced kinetic-rate constant (k/k(0)) method, where k and k(0) are the folding (unfolding) rate constants in the mutant and the wild-type forms, respectively, of a protein, has been applied to obtain qualitative information about structure in the transition state ensemble (TSE) of bovine pancreatic ribonuclease A (RNase A), which contains four native disulfide bonds. The method compares the folding (unfolding) kinetics of RNase A, with and without a covalent crosslink and tests whether the crosslinked residues are associated in the folding (unfolding) transition state (TS) of the noncrosslinked version. To confirm that the fifth disulfide bond has not introduced a significant structural perturbation, we solved the crystal structure of the V43C-R85C mutant to 1.6 A resolution. Our findings suggest that residues Val43 and Arg85 are not associated, and that residues Ala4 and Val118 may form nonnative contacts, in the folding (unfolding) TSE of RNase A.
一种先前引入的动力学速率常数(k/k(0))方法,其中k和k(0)分别是蛋白质突变体和野生型形式的折叠(去折叠)速率常数,已被应用于获取有关牛胰核糖核酸酶A(RNase A)过渡态系综(TSE)结构的定性信息,该酶含有四个天然二硫键。该方法比较了有无共价交联时RNase A的折叠(去折叠)动力学,并测试交联残基在非交联版本的折叠(去折叠)过渡态(TS)中是否相关联。为了确认第五个二硫键没有引入显著的结构扰动,我们解析了V43C-R85C突变体的晶体结构,分辨率达到1.6 Å。我们的研究结果表明,在RNase A的折叠(去折叠)TSE中,Val43和Arg85残基不相关联,而Ala4和Val118残基可能形成非天然接触。