Interactions of the major cold shock protein of Bacillus subtilis CspB with single-stranded DNA templates of different base composition
Interactions of the major cold shock protein of Bacillus subtilis CspB with single-stranded DNA templates of different base composition
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DOI：
10.1074/jbc.274.47.33601
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发表时间：
1999-11-19
影响因子：
4.8
通讯作者：
Makhatadze, GI
中科院分区：
文献类型：
--
作者：
Lopez, MM;Yutani, K;Makhatadze, GI
CspB是枯草芽孢杆菌的一种小的酸性蛋白，其诱导响应于冷休克而显著增加。虽然CspB的确切功能作用尚不清楚，但已证明该蛋白质结合单链脱氧核酸（ssDNA）。我们通过分析CspB与模型寡脱氧核苷酸相互作用的热力学，解决了碱基组成对CspB与ssDNA结合的影响的问题。结合四种不同的技术，荧光光谱，凝胶迁移率测定，等温滴定量热法和分析超离心，使我们能够表明：1）CspB可以优先结合聚嘧啶，而不是聚嘌呤ssDNA模板; 2）与基于T的ssDNA模板的结合以高亲和力发生（K-d（25 ℃）约为42 nM）并且是盐非依赖性的，而CspB与基于C的ssDNA模板的结合是强烈盐依赖性的（在IM NaCl下未观察到结合），表明相互作用中涉及大的静电分量; 3）在结合时，每个CspB覆盖基于T的ssDNA上的6-7个胸腺嘧啶碱基的延伸; CspB与T-ssDNA模板的结合是由构象驱动的，表明CspB上的芳香侧链与胸腺嘧啶碱基之间可能存在相互作用，并讨论了这些结果对CspB在细菌冷休克反应中功能作用的意义。
CspB is a small acidic protein of Bacillus subtilis, the induction of which is increased dramatically in response to cold shock. Although the exact functional role of CspB is unknown, it has been demonstrated that this protein binds single-stranded deoxynucleic acids (ssDNA). We addressed the question of the effect of base composition on the CspB binding to ssDNA by analyzing the thermodynamics of CspB interactions with model oligodeoxynucleotides. Combinations of four different techniques, fluorescence spectroscopy, gel shift mobility assays, isothermal titration calorimetry, and analytical ultracentrifugation, allowed us to show that: 1) CspB can preferentially bind poly-pyrimidine but not poly-purine ssDNA templates; 2) binding to T-based ssDNA template occurs with high affinity (K-d(25 degrees C) approximate to 42 nM) and is salt-independent, whereas binding of CspB to C-based ssDNA template is strongly salt-dependent (no binding is observed at 1 M NaCl), indicating large electrostatic component involved in the interactions; 3) upon binding each CspB covers a stretch of 6-7 thymine bases on T-based ssDNA; and 4) the binding of CspB to T-based ssDNA template is enthalpically driven, indicating the possible involvement of interactions between aromatic side chains on the protein with the thymine bases, The significance of these results with respect to the functional role of CspB in the bacterial cold shock response is discussed.