Ackerman Michael S, Shortle David
Department of Biological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
Biochemistry. 2002 Nov 19;41(46):13791-7. doi: 10.1021/bi020511t.
A nativelike low-resolution structure has been shown to persist in the Delta 131 Delta denatured fragment of staphylococcal nuclease, even in the presence of 8 M urea. In this report, the physical-chemical basis of this structure is addressed by monitoring changes in structure reflected in residual dipolar couplings and diffusion coefficients as a function of changes in amino acid sequence. Ten large hydrophobic residues, previously shown to play dominant roles in the stability of the native state, are replaced with polar residues of similar shape. Modest increases in the Stokes radius determined by NMR methods result from replacement of five isoleucine/valine residues with threonine, one leucine with glutamine, and oxidation of four methionines to the sulfoxides. Yet in the presence of all ten hydrophobic to polar substitutions and 8 M urea, the NMR signature of a native-like topology is still largely intact. In addition, removal of 30 residues from either the N-terminus (which deletes a three-strand beta meander) or C-terminus (a long extended segment and the final alpha helix) produces only very small changes in long-range structure. These data indicate that both the general shape of the denatured state and the angular relationships of individual bond angles to the axes describing the spatial distribution of the protein chain are insensitive to large changes in the amino acid sequence, a finding consistent with the conclusion that the long-range structure of denatured proteins is encoded primarily by local steric interactions between side chains and the polypeptide backbone.
已证明，即使在存在8 M尿素的情况下，葡萄球菌核酸酶的Delta 131 Delta变性片段中仍存在类似天然的低分辨率结构。在本报告中，通过监测残余偶极耦合和扩散系数反映的结构变化作为氨基酸序列变化的函数，探讨了这种结构的物理化学基础。先前已证明在天然状态稳定性中起主导作用的10个大的疏水残基被形状相似的极性残基取代。用苏氨酸取代5个异亮氨酸/缬氨酸残基、用谷氨酰胺取代1个亮氨酸以及将4个甲硫氨酸氧化为亚砜，通过核磁共振方法测定的斯托克斯半径会适度增加。然而，在存在所有10个疏水到极性取代以及8 M尿素的情况下，类似天然拓扑结构的核磁共振特征仍然基本完整。此外，从N端（删除一个三链β曲折）或C端（一个长的延伸片段和最后的α螺旋）去除30个残基，只会使远程结构产生非常小的变化。这些数据表明，变性状态的总体形状以及各个键角与描述蛋白质链空间分布的轴的角度关系对氨基酸序列的大变化不敏感，这一发现与变性蛋白质的远程结构主要由侧链与多肽主链之间的局部空间相互作用编码的结论一致。
