Flanagan J M, Kataoka M, Fujisawa T, Engelman D M
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06511.
Biochemistry. 1993 Oct 5;32(39):10359-70. doi: 10.1021/bi00090a011.
Deletion of 13 amino acids from the carboxyl terminus of staphylococcal nuclease (WTSNase delta) results in a denatured, partially unfolded molecule that lacks significant persistent secondary structure but is relatively compact and monomeric under physiological conditions [Shortle & Meeker (1989) Biochemistry 28, 936-944; Flanagan et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 748-752]. Because of these and other properties of the SNase delta polypeptide, it is a useful model system for investigating the conformation of the denatured state of a protein without using extreme temperature or solvent conditions. Moreover, since the modification is a carboxyl-terminal deletion, SNase delta may also resemble a transient state of the polypeptide chain as it emerges from a ribosome prior to its folding. In the present study, we have examined the sizes and conformations of mutated forms of SNase delta, using small-angle X-ray scattering and circular dichroism spectroscopy. Seven mutated forms were studied: four with single substitutions, two with double substitutions, and one triple substitution. When present in the full-length SNase, each of these mutated forms exhibited unusual behavior upon solvent or thermal denaturation. In the case of the truncated form (SNase delta), the small-angle scattering curves of the mutated forms fall into two classes: one resembling the scattering curve of compact native nuclease and the other having features consistent with those expected for an expanded coil-like polymer. In contrast, the scattering curve of WT SNase delta exhibits features intermediate between those observed for globular proteins and random polymers. The amino acid substitutions that gave rise to compact, native-like versions of SNase delta were all of the m--type (m-substitutions are predicted to decrease the size of the denatured state). Those which gave rise to versions of SNase delta that were more extended and coil-like than WT SNase delta were of the m+ type (m+ substitutions are predicted to increase the size of the denatured state). Estimates of the residual secondary structure present in WT SNase delta, as well as both the m+ and m-substituted versions of SNase delta, as determined by CD, suggest that the formation of secondary structure and compaction of the polypeptide chain occur concurrently. Our results show that single amino acid substitutions can radically alter the conformational distribution of a partially condensed polypeptide chain.(ABSTRACT TRUNCATED AT 400 WORDS)
从葡萄球菌核酸酶的羧基末端缺失13个氨基酸（WTSNase delta）会产生一个变性的、部分展开的分子，该分子缺乏显著的持续二级结构，但在生理条件下相对紧凑且为单体形式[肖特尔和米克（1989年）《生物化学》28卷，936 - 944页；弗拉纳根等人（1992年）《美国国家科学院院刊》89卷，748 - 752页]。由于SNase delta多肽的这些及其他特性，它是一个有用的模型系统，可用于在不使用极端温度或溶剂条件的情况下研究蛋白质变性状态的构象。此外，由于这种修饰是羧基末端缺失，SNase delta也可能类似于多肽链从核糖体中出来后在折叠之前的瞬态。在本研究中，我们使用小角X射线散射和圆二色光谱法研究了SNase delta突变形式的大小和构象。研究了七种突变形式：四种单取代、两种双取代和一种三取代。当存在于全长SNase中时，这些突变形式中的每一种在溶剂或热变性时都表现出异常行为。对于截短形式（SNase delta），突变形式的小角散射曲线分为两类：一类类似于紧密天然核酸酶的散射曲线，另一类具有与预期的扩展螺旋状聚合物一致的特征。相比之下，WT SNase delta的散射曲线呈现出介于球状蛋白质和无规聚合物所观察到的特征之间的特征。产生紧密的、类似天然形式的SNase delta氨基酸取代均为m型（m取代预计会减小变性状态的大小）。那些产生比WT SNase delta更伸展且呈螺旋状的SNase delta形式的取代为m +型（m +取代预计会增加变性状态的大小）。通过CD测定的WT SNase delta以及m +和m取代的SNase delta版本中存在的残余二级结构估计表明，二级结构的形成和多肽链的压缩同时发生。我们结果表明，单个氨基酸取代可从根本上改变部分浓缩多肽链的构象分布。（摘要截于400字）
