Wang Y, Shortle D
Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
Biochemistry. 1995 Dec 12;34(49):15895-905. doi: 10.1021/bi00049a004.
In a previous report [Alexandrescu, A. T., Abeygunawardana, C., & Shortle, D. (1994) Biochemistry 33, 1063-1072], NMR methods were used to characterize the residual structure in delta 131 delta, a large fragment of staphylococcal nuclease that serves as a model denatured state under nondenaturing conditions. On the basis of a large number of missing amide protons for the residues that form a three-strand antiparallel beta sheet in the native state, it was concluded that this beta meander may be highly populated in delta 131 delta, with severe line broadening due to relatively slow exchange between different conformational states. In the present report, results from circular dichroism spectroscopy and NMR spectroscopy indicate strands beta 2-beta 3 form a beta hairpin at urea concentrations below 6 M. Amide proton resonances from several hydrophobic residues adjacent to this beta hairpin disappear in concert with all of the beta 2-beta 3 residues, suggesting a local, non-native hydrophobic interaction may help stabilize the beta hairpin. At concentrations below 3 M, all amide resonances from strand beta 1 in delta 131 delta also disappear, suggesting that beta 1 may combine with the beta 2-beta 3 hairpin to form a native-like beta meander. In addition, the hydrophobic helix alpha 2 decreases from approximately 30% population in 0 M urea to approximately 10%-15% at 6 M urea, whereas helix alpha 1 goes from 10%-15% populated in 0 M urea to undetectable in 6 M urea. Characterization of a second, distinctly different denatured state, WT nuclease at pH 3.0 and low salt, reveals that this low-density acid-denatured state is structurally similar to delta 131 delta at low concentrations of urea. From these and previously published data, a tenative equilibrium folding pathway can be constructed for staphylococcal nuclease which describes the relative strengths and interdependencies of the chain-chain interactions involved in forming the native state.
在之前的一份报告中[亚历山德雷斯库，A.T.，阿贝古纳瓦德纳，C.，&肖特尔，D.（1994年）《生物化学》33卷，第1063 - 1072页]，核磁共振方法被用于表征δ131δ中的残余结构，δ131δ是葡萄球菌核酸酶的一个大片段，在非变性条件下作为一种模型变性状态。基于天然状态下形成三链反平行β折叠片的残基有大量缺失的酰胺质子，得出结论，这种β曲折结构在δ131δ中可能高度存在，由于不同构象状态之间相对缓慢的交换导致谱线严重展宽。在本报告中，圆二色光谱和核磁共振光谱的结果表明，在尿素浓度低于6 M时，β2 - β3链形成一个β发夹结构。与该β发夹结构相邻的几个疏水残基的酰胺质子共振与所有β2 - β3残基一起消失，表明局部的非天然疏水相互作用可能有助于稳定β发夹结构。在浓度低于3 M时，δ131δ中β1链的所有酰胺共振也消失，表明β1可能与β2 - β3发夹结构结合形成类似天然的β曲折结构。此外，疏水螺旋α2在0 M尿素中的占有率从约30%降至6 M尿素中的约10% - 15%，而螺旋α1在0 M尿素中的占有率从10% - 15%降至6 M尿素中无法检测到。对第二种明显不同的变性状态，即pH 3.0和低盐条件下的野生型核酸酶的表征表明，这种低密度酸变性状态在结构上与低浓度尿素下的δ131δ相似。根据这些以及之前发表的数据，可以构建一个葡萄球菌核酸酶的初步平衡折叠途径，该途径描述了形成天然状态过程中链 - 链相互作用的相对强度和相互依赖性。
