MOLECULAR DYNAMICS SIMULATIONS OF SITE-SPECIFIC PROTEIN-DNA INTERACTIONS: CONFI

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 特定DNA序列的蛋白质识别是许多正常和疾病相关过程的核心，包括基因表达及其调控和遗传重组。为了了解生物物理原理，管理识别特异性蛋白质-DNA相互作用，我们研究了三种限制性内切酶（EcoRI，BamHI和EcoRV）与它们的DNA识别位点的相互作用，使用热力学，动力学，光谱（NMR，EPR和荧光），遗传学和计算机模拟。主要关注蛋白质-DNA复合物的动力学特性，包括蛋白质和DNA中依赖于结合的构象转变以及复合物中的构象振动波动。我们对这些系统的各种信息的巨大积累使我们能够提出尖锐的计算问题，并将计算结果与实验数据进行基准测试。该建议包括以下计算目标：1。利用计算机模拟研究一种“混杂”突变型EcoRI内切酶与其DNA识别位点的相互作用。我们将首先测试MD模拟（使用AMBER 8套件，使用明确的溶剂并通过粒子-网格Ewald处理长程相互作用）是否可以重现突变体和野生型复合物之间相对细微的差异，如X射线晶体学所确定的，为我们缺乏实验结构的突变体的模拟铺平道路。然后，我们将使用MD模拟来研究突变体复合物的动力学，特别注意界面水分子的作用，包裹DNA的蛋白质“臂”的运动，以及复合物中可能的协同运动。这些研究将包括从MD轨迹计算Debye-Waller B因子，并与实验B因子进行比较。最后，我们将使用野生型和突变型复合物的数据进行计算时间平均晶体学精修（TACR），以研究物理上合理的并受实验衍射数据定义的包络约束的分子和溶剂运动。这些研究将使用GROMOS软件包进行TACR的MD部分。2.使用MD模拟（全显式溶剂），以研究如何构象动力学和EcoRI-DNA复合物中的分子畸变的影响，立体特异性甲基膦酸酯取代在两个位置的GAATTC识别位点，我们有广泛的生化数据。我们将使用这样的模拟来研究这些衍生物如何导致（a）催化所需的精确水中继中断;（B）侧链构象的微妙但关键的改变;（c）防止Mg 2+辅因子结合到活性位点。这些研究应该产生新的见解的作用，活性位点的水结构和DNA的扭曲的催化机制。3.利用分子动力学模拟研究BamHI-DNA复合物活性位点的静电排斥（分子张力的一种形式）的后果。实验数据表明，该活性位点中的酸性侧链簇具有异常高的pKa，E111侧链上的电荷是关键的中央控制器，并且应变的后果包括在复合物中增加的分子运动（加宽的动态分布）。我们将首先使用计算机重建和MD模拟（全显式溶剂）来产生野生型BamHI-DNA复合物的改进模型，因为我们的生物化学数据显示晶体结构缺乏必要的DNA元素，并且由于包装力而受到扭曲。然后，我们将在野生型蛋白质和突变蛋白质E111 A和D94 A的背景下测试E111和D94的质子化的效果，检查以下问题：（a）菌株的后果是否严格局部（例如，旋转异构体松弛）或扩展到复合体的更广泛区域？(b)在应变复合物中增加的构象振动运动主要是在小区域内的大运动还是在较大区域内的小运动？(c)蛋白质和DNA是否进行协调运动？我们将使用一个大的显式溶剂壳，这使我们能够明确地处理蛋白质的灵活性和介电弛豫，最后，我们将使用分子动力学自由能模拟来计算E111在野生型和D94 A的背景下的pKa位移。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Protein recognition of specific DNA sequences lies at the heart of many normal and disease-related processes, including gene expression and its regulation and genetic recombination. To understand the biophysical principles that govern recognition specificity in protein-DNA interactions, we study the interactions of three restriction endonucleases (EcoRI, BamHI and EcoRV) with their DNA recognition sites, using thermodynamics, kinetics, spectroscopy (NMR, EPR and fluorescence), genetics and computational simulations. Major attention is given to dynamic characteristics of protein-DNA complexes, including binding-dependent conformational transitions in proteins and DNA and conformational-vibrational fluctuations in the complexes. Our enormous accumulation of diverse information on these systems places us in position to pose sharply focused computational questions and to benchmark the computational results against experimental data. This proposal includes the following computational objectives: 1. To use computational simulations to study the interactions of a "promiscuous" mutant EcoRI endonuclease with its DNA recognition site. We will first test if MD simulations (using the AMBER8 suite, with explicit solvent and treatment of long-range interactions by particle-mesh Ewald) can reproduce the relatively subtle differences between mutant and wild-type complexes as determined by x-ray crystallography, to pave the way for simulations of the mutants for which we lack experimental structures. We will then use MD simulations to study the dynamics of the mutant complex, with particular attention to the roles of interfacial water molecules, motions of the protein "arms" that enfold the DNA, and possibly concerted motions in the complex. These studies will include calculation of Debye-Waller B-factors from the MD trajectories, and comparison with the experimental B-factors. Finally, we will do computational time-averaging crystallographic refinement (TACR) using the data on both wild-type and mutant complexes, to study molecular and solvent motions that are physically reasonable and constrained to the envelope defined by the experimental diffraction data. These studies will use the GROMOS package for the MD portion of the TACR. 2. To use MD simulations (with full explicit solvent) to study how conformational dynamics and molecular distortions in the EcoRI-DNA complex are affected by stereospecific methylphosphonate substitutions at two positions of the GAATTC recognition site, for which we have extensive biochemical data. We will use such simulations to study how these derivatives cause (a) disruption of the precise water relay required for catalysis; (b) subtle but crucial alterations in sidechain conformations; (c) prevention of Mg2+ cofactor binding to the active site. These studies should yield new insight into the role of active-site water structure and DNA distortion in the catalytic mechanism. 3. To use MD simulations to study the consequences of electrostatic repulsion (a form of molecular strain) in the active site of the BamHI-DNA complex. Experimental data indicate that the cluster of acidic sidechains in this active site have abnormally high pKa's, that the charge on the E111 sidechain is the critical central controller, and that the consequences of strain include increased molecular motion (broadened dynamic distribution) in the complex. We will first use in silico rebuilding and MD simulation (full explicit solvent) to produce an improved model of the wild-type BamHI-DNA complex, since our biochemical data show the crystal structure lacks essential DNA elements and suffers from distortions due to packing forces. We will then test the effect of protonation of E111 and D94 in the context of wild-type protein and mutant proteins E111A and D94A, examining the following issues: (a) Are the consequences of strain strictly local (e.g., rotamer relaxation) or extended over wider areas of the complex? (b) Are the increased conformational-vibrational motions in strained complexes primarily large motions in a small region or smaller motions over a larger region? (c) Do the protein and DNA undergo concerted motions? We will use a large explicit solvent shell, which allows us to treat protein flexibility and dielectric relaxation explicitly, Finally, we will use molecular dynamics free energy simulations to calculate the pKa shifts of E111 in the context of wild-type and D94A.