Recognition reactions between macromolecules

大分子之间的识别反应

• 批准号: 7734810
• 负责人: Donald Rau
• 金额: $ 26.99万
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7734810
• 关键词: Affinity; Area; Binding; Binding Proteins; Biological Assay; Biological Models; Buffers; Capillary Electrophoresis; Cell physiology; Cleaved cell; Competitive Binding; Complex; Condition; Coupling; Crowding; DNA; DNA Binding; DNA Restriction Enzymes; DNA Sequence; DNA-Protein Interaction; Dependence; Dissociation; Electrophoresis; Enzymes; Equilibrium; Exclusion; Explosion; Gel; Gel Chromatography; Goals; Half-Life; Hour; Hydration status; Individual; Kinetics; Label; Laboratories; Literature; Measurement; Measures; Methods; Nucleic Acids; Numbers; Oligonucleotides; Osmotic Pressure; Play; Protein Binding; Proteins; Protocols documentation; Rate; Reaction; Reaction Time; Relative (related person); Reporting; Roentgen Rays; Role; Sequence-Specific DNA Binding Protein; Solutions; Solvents; Specificity; Stress; Structure; Surface; Techniques; Thermodynamics; Time; Type II site-specific deoxyribonuclease; Water; Work; dimer; fluorophore; gel mobility shift assay; macromolecule; migration; novel; nuclease; polyacrylamide gels; polymerization; protein structure; research study; solute; tool; triethylene glycol; trimethyloxamine

We have focused on DNA complexes of restriction endonucleases in particular. Beyond their unparalleled importance as tools for the analysis and manipulation of DNA, restriction enzymes have proven remarkable model systems for studying numerous aspects of protein-nucleic acid interactions. These proteins combine high binding strength and extraordinary sequence specificity. Work is currently in progress on the type II restriction enzyme, EcoRV. Typically restriction endonucleases can distinguish between specific recognition and nonspecific DNA sequences quite efficiently. At present, however, results in literature suggest a very low sequence stringency for EcoRV. The majority of studies show only weak selectivity, below 10 for the relative specific-nonspecific competitive binding constant. Only one group has reported value of 120 at the same experimental conditions. X-ray crystal structures are available for both cognate and non-cognate EcoRV complexes. The interface of the specific complex is essentially anhydrous with many direct DNA-protein interactions and is very different from the non-cognate complex that has a large water filled gap at the protein-DNA interface. The substantial difference in structure would suggest a large difference in binding energy as is true for other type II restriction enzymes. We have applied our unique perspective and experimental tools to reexamine this nuclease. The novel self-cleavage assay we developed recently is broadly applicable to measuring DNA-protein interactions, particularly DNA binding of restriction endonucleases. This solution technique uses the cleavage reaction of restriction endonucleases to measure sensitively their binding to DNA. It has same sensitivity as gel mobility shift assay without its limitations. Additionally the self-cleavage assay does not require labeling DNA or protein with fluorophores that could perturb binding and that are typically sensitive to buffer conditions. We have shown that the self-cleavage assay can be used to quantitate EcoRV-DNA binding. After adding cleavage mix containing Mg2+, high enough concentrations of competing oligonucleotide that contains the recognition sequence, and neutral osmolytes, e.g., triethylene glycol or TMAO, to the pre-formed complex, only DNA fragments with initially bound enzyme are cleaved. Equilibrium measurements are meaningful only after it is known that the reaction time is sufficiently long to reach equilibrium. It is not unusual for proteins that have a high affinity for DNA to dissociate quite slowly from its specific sequences with half-life times measured in minutes and even hours. Association rate constants are usually much faster. Surprisingly, the association kinetics of EcoRV shows at least two components; one that is quite fast as is typical for specific binding. The other is unusually slow with a half-life time of about 20 min. This is far different from our previous observations with EcoRI. These results and the higher order complexes seen with the gel mobility shift assay suggest that EcoRV can form tetramers on DNA. We do not know if tetramers are preformed in solution or dimers meet on the DNA. We can distinguish the two by changing the relative and absolute concentrations of protein and DNA. The slow component of the association kinetics indicates that at least 2 hours incubation is necessary to reach equilibrium. The unusual on-rate kinetics for EcoRV could explain the puzzling literature results. It would be easy to underestimate amount of bound protein (and consequently binding constant) if the incubation time was not long enough to reach equilibrium. We have shown that the relative specific-nonspecific binding constant for EcoRV depends on water activity with a significant increase in binding specificity with increase of triethylene glycol concentration. We can determine the difference in sequestered water between the complexes through the dependence of the relative specific-nonspecific binding constant on solution osmotic pressure. Our preliminary estimation is that the nonspecific complex sequesters about 160 more water molecules than the specific complex. This value correlates quite well with the difference in volume of the interface cavities of specific and non-cognate complexes seen in the crystal structures. We have also estimated the relative specific-nonspecific binding constant as about 400, a value much larger than previously measured by others. As with other restriction nucleases, EcoRV specific sequence binding is stringent. We have also developed a method for stabilizing labile DNA-protein complexes for analysis by the gel mobility shift assay. Polyacrylamide gels stabilize DNA-protein or protein-protein complexes by a crowding or caging mechanism. Many nonspecific DNA-protein complexes, however, are weak enough that they do dissociate while electrophoresing giving smeared bands that are difficult to quantitate precisely. We find that adding osmolytes directly to the gel can further stabilize weak complexes. This project is a logical continuation of our previous work on trapping DNA-protein complexes. We now routinely use the stop-reaction protocol for all gel mobility shift assays. Since complex dissociation in the gel matrix is accompanied by a change in solvent accessible surface area (SASA), different osmolytes will have different efficacies depending on their extent of exclusion from the newly exposed surfaces. Experiments working with nonspecific complexes of EcoRI and BamHI show that triethylene glycol is particularly effective at inhibiting dissociation, does not interfere with normal gel polymerization, and does not significantly slow normal migration. Extension of this approach to other techniques for separating complex and free components as gel chromatography and capillary electrophoresis is straightforward. Work is now in progress to develop even better stabilizing conditions and fully eliminate in-gel dissociation. We intend to check variety of neutral solutes for their ability to stabilize weak DNA-protein in the gel.

我们特别关注限制性核酸内切酶的DNA复合物。除了作为DNA分析和操纵的工具的无与伦比的重要性外，限制酶还证明了非凡的模型系统，用于研究蛋白质核酸相互作用的许多方面。 这些蛋白质结合了高结合强度和非凡序列特异性。 目前正在进行II型限制酶ECORV的工作。通常，限制性内切酶可以非常有效地区分特定识别和非特异性DNA序列。然而，目前的文献结果表明，ECORV的序列严格度非常低。大多数研究仅显示弱选择性，相对非特异性竞争结合常数低于10。在相同的实验条件下，只有一组报告的值为120。 X射线晶体结构可用于同源和非同义Ecorv复合物。特定复合物的界面基本上是无水的，具有许多直接DNA-蛋白相互作用，并且与在蛋白质-DNA界面上具有较大水的间隙的非同名复合物截然不同。结构的实质差异将表明与其他II型限制酶一样，结合能有很大的差异。 我们已经应用了独特的视角和实验工具来重新检查此核酸酶。我们最近开发的新型自切解测定法广泛适用于测量DNA-蛋白质相互作用，尤其是限制性核酸内切酶的DNA结合。该溶液技术使用限制性核酸内切酶的切割反应来测量其与DNA的结合。它的灵敏度与凝胶移动性转移测定法相同，而无需限制。另外，自切除测定法不需要将DNA或蛋白质标记为荧光团，而荧光团可能会扰动结合并且通常对缓冲液条件敏感。我们已经表明，自切解测定可用于定量Ecorv-DNA结合。 在添加含有MG2+的裂解混合物后，足够高的竞争性寡核苷酸含有识别序列，中性的渗透液（例如，三乙二醇或TMAO）到预先形成的络合物中，仅带有最初结合酶的DNA片段。 仅在知道反应时间足够长以达到平衡之后，平衡测量才有意义。对于DNA具有较高亲和力的蛋白质，与其特定序列相结合的蛋白质并不罕见，并且在几分钟甚至数小时内测得的半衰期时间。关联率常数通常更快。令人惊讶的是，ECORV的关联动力学至少显示了两个组成部分。一个非常快的特定结合。另一个时间异常缓慢，半衰期约为20分钟。这与我们以前对ECORI的观察有很大不同。这些结果以及用凝胶迁移率转移测定法看到的高阶复合物表明ECORV可以在DNA上形成四聚体。我们不知道四聚体是在溶液中预先形成的还是在DNA上相遇的二聚体。我们可以通过改变蛋白质和DNA的相对浓度来区分两者。关联动力学的缓慢成分表明，至少需要2小时的孵育才能达到平衡。 ECORV的异常率动力学可以解释令人困惑的文献结果。如果孵育时间不足以达到平衡，则很容易低估限制的蛋白质（并因此结合常数）。 我们已经表明，ECORV的相对特异性非特异性结合常数取决于水的活性，而结合特异性随着三乙二醇浓度的增加而显着增加。我们可以通过相对特异性非特异性结合常数对溶液渗透压的依赖性来确定复合物之间的隔离水之间的差异。我们的初步估计是，非特异性复合物隔离比特定复合物多160个。该值与晶体结构中特定和非共同络合物的界面腔体积的差异非常相关。我们还估计相对特异性非特异性结合常数约为400，一个值比其他人先前测量的值大得多。与其他限制核酸酶一样，ECORV特定序列结合也很严格。 我们还开发了一种稳定不稳定DNA蛋白复合物的方法，用于通过凝胶迁移率转移测定法分析。聚丙烯酰胺凝胶通过拥挤或笼子机制稳定DNA-蛋白质或蛋白质 - 蛋白质复合物。然而，许多非特异性DNA蛋白复合物的弱较弱，以至于它们在电体的同时会分离，从而产生了很难精确定量的涂抹带。我们发现，将渗透液直接添加到凝胶中可以进一步稳定弱复合物。该项目是我们以前关于捕获DNA-蛋白质复合物的工作的逻辑延续。现在，我们常规地将停止反应协议用于所有凝胶移动性转移测定法。由于凝胶基质中的复杂解离伴随着溶剂可访问的表面积（SASA）的变化，因此不同的渗透压将具有不同的效率，具体取决于它们从新裸露的表面排除的程度。与ECORI和BAMHI非特异性复合物一起使用的实验表明，三乙二醇在抑制解离时特别有效，不会干扰正常的凝胶聚合，并且不会显着慢慢迁移。将这种方法扩展到将复合和游离成分分离为凝胶色谱和毛细管电泳的其他技术。 现在正在进行工作，以发展更好的稳定条件并完全消除凝胶内解离。我们打算检查各种中性溶质是否能够稳定凝胶中弱DNA-蛋白质的能力。