Trevino R J, Gliubich F, Berni R, Cianci M, Chirgwin J M, Zanotti G, Horowitz P M
Department of Biochemistry, the University of Texas Health Science Center, San Antonio, Texas 78284, USA.
J Biol Chem. 1999 May 14;274(20):13938-47. doi: 10.1074/jbc.274.20.13938.
The NH2-terminal sequence of rhodanese influences many of its properties, ranging from mitochondrial import to folding. Rhodanese truncated by >9 residues is degraded in Escherichia coli. Mutant enzymes with lesser truncations are recoverable and active, but they show altered active site reactivities (Trevino, R. J., Tsalkova, T., Dramer, G., Hardesty, B., Chirgwin, J. M., and Horowitz, P. M. (1998) J. Biol. Chem. 273, 27841-27847), suggesting that the NH2-terminal sequence stabilizes the overall structure. We tested aspects of the conformations of these shortened species. Intrinsic and probe fluorescence showed that truncation decreased stability and increased hydrophobic exposure, while near UV CD suggested altered tertiary structure. Under native conditions, truncated rhodanese bound to GroEL and was released and reactivated by adding ATP and GroES, suggesting equilibrium between native and non-native conformers. Furthermore, GroEL assisted folding of denatured mutants to the same extent as wild type, although at a reduced rate. X-ray crystallography showed that Delta1-7 crystallized isomorphously with wild type in polyethyleneglycol, and the structure was highly conserved. Thus, the missing NH2-terminal residues that contribute to global stability of the native structure in solution do not significantly alter contacts at the atomic level of the crystallized protein. The two-domain structure of rhodanese was not significantly altered by drastically different crystallization conditions or crystal packing suggesting rigidity of the native rhodanese domains and the stabilization of the interdomain interactions by the crystal environment. The results support a model in which loss of interactions near the rhodanese NH2 terminus does not distort the folded native structure but does facilitate the transition in solution to a molten globule state, which among other things, can interact with molecular chaperones.
硫氰酸酶的氨基末端序列影响其许多特性，从线粒体导入到折叠。被截短超过9个残基的硫氰酸酶在大肠杆菌中会被降解。截短程度较小的突变酶可回收且具有活性，但它们表现出活性位点反应性的改变（特雷维诺，R. J.，察尔科娃，T.，德拉默，G.，哈迪斯蒂，B.，奇尔文，J. M.，和霍洛维茨，P. M.（1998年）《生物化学杂志》273，27841 - 27847），这表明氨基末端序列稳定了整体结构。我们测试了这些缩短形式的构象方面。内在荧光和探针荧光表明截短降低了稳定性并增加了疏水暴露，而近紫外圆二色性表明三级结构发生了改变。在天然条件下，截短的硫氰酸酶与GroEL结合，并通过添加ATP和GroES而被释放并重新激活，这表明天然构象和非天然构象之间存在平衡。此外，GroEL辅助变性突变体折叠的程度与野生型相同，尽管速率有所降低。X射线晶体学表明，Δ1 - 7在聚乙二醇中与野生型同晶型结晶，且结构高度保守。因此，在溶液中对天然结构整体稳定性有贡献的缺失氨基末端残基，在结晶蛋白的原子水平上并未显著改变接触。硫氰酸酶的双结构域结构并未因截然不同的结晶条件或晶体堆积而发生显著改变，这表明天然硫氰酸酶结构域具有刚性，且晶体环境稳定了结构域间的相互作用。这些结果支持了一个模型，即硫氰酸酶氨基末端附近相互作用的丧失不会扭曲折叠的天然结构，但确实促进了溶液中向熔球态的转变，熔球态除其他作用外，还能与分子伴侣相互作用。
