Sequence specific DNA protein interactions

 描述（由申请人提供）：位点特异性DNA-蛋白质相互作用在生物学的各个方面发挥着核心作用。该提案侧重于蛋白质-DNA复合物的动力学，建立内部动力学与亲和力和特异性测量之间的联系。蛋白质与DNA上邻近位点的结合通常是协同的，并且DNA本身可以介导协同性。因此，蛋白质和DNA的动力学可能会影响相邻的相互作用。利用EcoRV内切酶与DNA的高度特异性相互作用作为模型，我们研究了两种动力学探针：（a）识别位点周围的DNA序列背景，这可能影响DNA弯曲的能量成本和整个复合物中位置的优化;（B）Lu 3+离子，其作为Mg 2+替代物结合在活性位点中并中和蛋白质和DNA之间的静电排斥。这两种探针对结合都有很大的影响（1900倍范围的背景，22,000倍的金属离子添加），并显着增强特异性。两者都对与EcoRV-DNA结合相关的热容变化有深远的影响。β C°P是特异性蛋白质-DNA相互作用的关键标志，因为特异性复合物的热容（β C°P<<0）会大幅下降，而非特异性复合物则不会。因此，它反映了蛋白质-DNA界面的拟合优度。侧翼三重变异不会引起EcoRV-DNA复合物的结构或表面去溶剂化程度的变化，从而大大简化了分析。热力学数据表明，对β C°P的影响可能是由于蛋白质-DNA复合物动力学的差异，并且太大而不仅仅是局部的;甲基弛豫的初步NMR研究支持这一点。我们的中心假设是，这两个因素都调节了复合体中的动力自由度，并且受限动力学对负的CO2 C°P做出了主要贡献。 对EcoRV-DNA复合物的热力学研究将测量自由能、焓、熵和Δ C°P的探针依赖性变化。核磁共振动力学研究将确定是否动态变化，由于DNA的背景下，离子和活性位点突变映射到不同的区域。甲基碳弛豫和交换色散测量将用于表征蛋白质的局部ps-ns和µs-ms运动。将使用DNA中31 P、19 F、15 N和13 C基团的弛豫和交换分散来评估DNA动力学。这包括一种新的使用DNA硫代磷酸酯的NMR动力学研究。广义NMR有序参数及其温度依赖性将用于将动力学与Δ S°和Δ C°P的热力学差异联系起来。因此，本项目将阐明金属离子如何限制构象波动 对于那些处于催化过渡态的酶，催化过程中的一个潜在的关键因素是各种各样的磷酸二酯酶和其他金属酶。这对于设计针对感染性、遗传性或代谢性疾病的关键酶的药物具有重要意义。

 DESCRIPTION (provided by applicant): Site-specific DNA-protein interactions play a central role in all aspects of biology. This proposal focuses on the dynamics in protein-DNA complexes, forging links between the internal dynamics and measures of affinity and specificity. Binding of proteins to proximal sites on DNA is often cooperative, and the DNA itself may mediate cooperativity. Thus, the dynamics of both protein and DNA may influence neighboring interactions. Using the highly specific interaction of EcoRV endonuclease with DNA as a model, we investigate two probes of the dynamics: (a) DNA sequence context around the recognition site, which likely affects the energetic cost of DNA bending and the optimization of positions throughout the complex; (b) Lu3+ ions that bind in the active sites as Mg2+ surrogates and neutralize electrostatic repulsion between protein and DNA. Both probes have large effects on binding (1900-fold range for context, 22,000-fold for addition of metal ions) and markedly enhance specificity. Both have profound effects on the heat capacity change C°P associated with EcoRV-DNA binding. C°P is a key hallmark of specific protein-DNA interactions, because a large decrease in the heat capacity (C°P<<0) occurs for specific complexes, but not nonspecific complexes. It thus reflects goodness of fit in a protein-DNA interface. Flanking triple variation does not cause changes in the structure of the EcoRV-DNA complex or the degree of surface desolvation, thus greatly simplifying the analysis. Thermodynamic data suggest that the effects on C°P are likely due to differences in dynamics of the protein-DNA complex and are too large to be merely local; preliminary NMR studies of methyl relaxation support this. Our central hypotheses are that both these factors tune dynamic freedom in the complex and that restricted dynamics make a dominant contribution to a negative C°P. Thermodynamic studies on EcoRV-DNA complexes will measure probe-dependent variation in free energy, enthalpy, entropy and C°P. NMR dynamics studies will determine whether dynamic changes due to DNA context, ions and active-site mutations map to distinct regions. Methyl carbon relaxation and exchange dispersion measurements will be used to characterize local ps-ns and µs-ms motions of the protein. DNA dynamics will be assessed using relaxation and exchange dispersion of 31P, 19F, 15N and 13C groups in DNA. This includes a novel use of DNA phosphorothioates for NMR dynamics studies. Generalized NMR order parameters and their temperature-dependences will be used to relate dynamics to thermodynamic differences in S° and C°P. This project will thus illuminate how metal ions constrain conformational fluctuations to those on the path to the catalytic transition state, a potentially critical factor in catalysis y a wide variety of phosphodiesterases and other metalloenzymes. This can have important implications for design of drugs targeting key enzymes of infectious, genetic or metabolic diseases.