Creighton T E, Shortle D
European Molecular Biology Laboratory, Heidelberg, Germany.
J Mol Biol. 1994 Oct 7;242(5):670-82. doi: 10.1006/jmbi.1994.1616.
The denatured state of staphylococcal (staph) nuclease has been shown to be altered by mutations, which complicates studies of stability of the folded state and may be important for the folding process. The relative hydrodynamic volumes as a function of urea concentration of staph nuclease, and of mutant forms in which the native state was substantially destabilized, have been examined by urea gradient gel electrophoresis. The native conformation is unique in its stabilization by specific ligands, which made it possible to distinguish between intermediate electrophoretic mobilities resulting from a partly populated native conformation and from other partly folded conformations. Four distinct conformational states were observed; two native-like: (1) the native, with and without bound ligands, and (2) an altered native-ligand complex at high urea concentrations; plus two denatured states: (3) a partly folded compact conformation that unfolded co-operatively and was sensitive to mutation of residues in the beta-barrel portion of the protein, and (4) the unfolded state, which appeared from its hydrodynamic volume to be as unfolded as reduced, alkylated ribonuclease A, even at very low urea concentrations, and was not altered by single mutations. The effects of mutations on the denatured state of staph nuclease may be due to the occurrence of the partly folded conformation (3).
已表明葡萄球菌核酸酶的变性状态会因突变而改变，这使折叠态稳定性的研究变得复杂，且可能对折叠过程很重要。通过尿素梯度凝胶电泳研究了葡萄球菌核酸酶及其天然状态大幅去稳定化的突变体形式的相对流体力学体积随尿素浓度的变化。天然构象在通过特定配体进行稳定方面具有独特性，这使得能够区分由部分占据的天然构象和其他部分折叠构象导致的中间电泳迁移率。观察到四种不同的构象状态；两种类似天然态：（1）有和没有结合配体的天然态，以及（2）在高尿素浓度下改变的天然 - 配体复合物；加上两种变性态：（3）一种部分折叠的紧密构象，其协同展开且对蛋白质β桶部分的残基突变敏感，以及（4）未折叠态，从其流体力学体积来看，即使在非常低的尿素浓度下也与还原的、烷基化的核糖核酸酶A一样未折叠，且不受单个突变的影响。突变对葡萄球菌核酸酶变性态的影响可能归因于部分折叠构象（3）的出现。
