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

位点特异性蛋白质-DNA 相互作用的分子动力学模拟：CONFI

• 批准号: 7601395
• 负责人: LINDA JEN-JACOBSON
• 金额: $ 0.03万
• 依托单位: CARNEGIE-MELLON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-08-01 至 2008-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7601395
• 关键词: Active Sites; Affect; Area; Attention; Benchmarking; Binding; Biochemical; Catalysis; Characteristics; Charge; Complement Factor B; Complex; Computer Retrieval of Information on Scientific Projects Database; Computer Simulation; Crystallography; DNA; DNA Restriction Enzymes; DNA Sequence; DNA Structure; DNA-Protein Interaction; Data; Deoxyribonuclease EcoRI; Disease; Disruption; Electrostatics; Elements; Fluorescence; Free Energy; Funding; Gene Expression; Genetic; Genetic Recombination; Grant; Heart; Institution; Kinetics; Modeling; Molecular; Molecular Conformation; Motion; NMR Spectroscopy; Pliability; Positioning Attribute; Prevention; Process; Proteins; Range; Regulation; Relaxation; Research; Research Personnel; Resources; Role; Site; Solvents; Source; Specificity; Structure; System; Testing; Thermodynamics; Time; United States National Institutes of Health; Upper arm; Water; cofactor; conformational conversion; improved; insight; interfacial; methylphosphonate; molecular dynamics; mutant; particle; protonation; simulation

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.

该副本是利用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这不一定是调查员的机构。 蛋白质对特定DNA序列的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节和遗传重组。为了了解控制蛋白质-DNA相互作用识别特异性的生物物理原理，我们研究了三个限制性内切酶（ECORI，BAMHI和ECORV）与其DNA识别位点的相互作用，使用热力学，动力学，光学，光谱，光谱，NMR，EPR和荧光和植物和计算模拟。对蛋白质-DNA复合物的动态特征给予了主要关注，包括蛋白质和DNA中结合依赖性构象转变，以及络合物中构象 - 振动的波动。我们在这些系统上大量的各种信息的积累使我们处于适当的关注计算问题上，并根据实验数据对计算结果进行基准测试。该建议包括以下计算目标：1。使用计算模拟研究“混杂”突变的ECORI核酸内切酶与其DNA识别位点的相互作用。我们将首先测试MD模拟（使用Amber8 Suite，具有明显的溶剂和粒子网埃瓦尔德的长距离相互作用的处理）可以重现突变体和野生型复合物之间通过X射线晶体学确定的相对细微的差异，从而为我们缺乏实验性结构的突变体的模拟方式铺平了。然后，我们将使用MD模拟研究突变体复合物的动力学，特别注意界面水分子的作用，蛋白质“臂”的运动，使DNA含有DNA，并可能在该综合体中进行协同运动。这些研究将包括从MD轨迹的Debye-Waller B因子计算，以及与实验B因子进行比较。最后，我们将使用野生型和突变体复合物的数据进行计算时间平衡的晶体学改进（TACR），以研究物理上合理的分子和溶剂运动，并限制在实验性衍射数据定义的信封上。这些研究将使用Gromos软件包作为TACR的MD部分。 2。要使用MD模拟（具有完全明确的溶剂）来研究Ecori-DNA复合物中的构象动力学和分子畸变如何受到GAATTC识别位点两个位置的立体甲基膦酸酯取代的影响，为此我们拥有广泛的生物化学数据。我们将使用此类模拟来研究这些衍生物如何引起（a）破坏催化所需的精确水中继； （b）Sidechain构象的微妙但至关重要的改变； （c）预防MG2+辅因子与活性位点结合。这些研究应对活性位点水结构和DNA失真在催化机制中的作用产生新的见解。 3。使用MD模拟研究BAMHI-DNA复合物的活性位点中静电排斥的后果（一种分子菌株形式）。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，即E111 Sidechain上的电荷是关键的中央控制器，并且菌株的后果包括复合物中分子运动的增加（扩展动态分布）。我们将首先用于计算机重建和MD模拟（完全显式溶剂），以产生改进的野生型BAMHI-DNA复合物的模型，因为我们的生化数据表明，晶体结构缺乏基本的DNA元素，并且由于包装力引起的扭曲而受到扭曲。然后，我们将在野生型蛋白质和突变蛋白E111A和D94A的背景下测试E111和D94的质子化的作用，研究以下问题：（a）严格局部局部（例如，旋转型）的后果（例如，旋转型）或扩展到更广泛的区域？ （b）在较大区域的小区域或较小的运动中，应变复合物中构象 - 振动的运动是否增加？ （c）蛋白质和DNA是否经历了一致的运动？我们将使用大型的显式溶剂壳，这使我们能够明确处理蛋白质的柔韧性和介电弛豫，最后，我们将使用分子动力学自由能模拟来计算野生型和D94A的情况下计算E111的PKA偏移。