Recognition reactions between macromolecules

大分子之间的识别反应

• 批准号: 8149342
• 负责人: Donald Rau
• 金额: $ 36.28万
• 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/8149342
• 关键词: Reaction; macromolecule

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. We take advantage of the unique experimental tools we have developed to investigate the coupling of structure, thermodynamics and function of these complexes from a fresh perspective. We are currently investigating DNA complexes of the type II restriction enzyme, EcoRV. Typically restriction endonucleases can distinguish between specific recognition and nonspecific DNA sequences quite efficiently in the absence of divalent metal co-factors that are required for cleavage. At present, however, results in literature suggest that EcoRV has unusually low sequence stringency. The majority of studies performed at pH 7.5, that is optimal for the EcoRV cleavage activity, have found less than a 10-fold difference between EcoRV binding constants to the specific and nonspecific DNA sequences in the absence of divalent ions. There is, however, one measurement in the literature that contradicts this lack of recognition stringency. Additionally, X-ray crystal structures for specific and non-cognate DNA-EcoRV complexes solved in the absence of metal co-factors are noticeably different. 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 suggesting significant differences in hydration and in binding free energies between two complexes. The biochemical techniques used to measure EcoRV-DNA binding were primarily the gel mobility shift assay and a variety of fluorescent techniques, all of which are prone to artifacts. We have applied a self-cleavage assay, developed previously by us, to measure EcoRV-DNA binding in solution. This technique monitors only enzymatically competent complexes of the endonuclease. It does not have the limitations of gel mobility shift assay while providing same level of sensitivity. Equilibrium measurements require knowledge of association rates, in particular. We found that the EcoRV has quite unusual kinetics of specific complex formation in the absence of divalent ions that was not observed for EcoRI. A significant fraction of the total enzyme, 45%, forms enzymatically competent complexes unusually slowly, especially at pH 7.6. This novel result can be explained by a very slow transition between two conformations of the free enzyme in solution. The equilibrium distribution of the slowly and quickly associating protein structures and their exchange kinetics may depend on many parameters including pH, salt, osmolytes, and divalent cations. The slow rate of complex formation could explain the lack of specificity reported by others. We have measured the ratio of specific and nonspecific binding constants using the self-cleavage assay, providing the long incubation times necessary to achieve equilibrium. At pH 7.6, the binding constant to a 310 bp fragment is 60-fold higher than binding constant to a 30-bp nonspecific oligonucleotide. This is about an order of magnitude larger than has been typically observed. The relative specific-nonspecific binding constant, Knsp-sp, increases strongly with decreasing pH and with increasing neutral osmolyte concentration. The osmotic pressure dependence of the relative binding constant is only weakly sensitive to pH indicating that the structures of the specific and nonspecific complexes as reflected by differences in sequestered water change minimally with pH. The large osmotic dependence observed for the Knsp-sp means that measurement of protein-DNA specificity in dilute solution cannot be directly applied to binding in the crowded environment of the cell. In addition to divalent ions, water activity and pH are two key parameters that strongly modulate binding specificity of the EcoRV. The observation of at least two kinetics components in association indicates that EcoRV is an allosteric protein with at least two conformations. Allosterism is now recognized as important concept for DNA-protein complexes, offering an additional level of control over binding and activity. The recognition specificity or activity of DNA binding proteins can be modulated by ligands or proteins that bind to one allosteric conformation in preference to others. We are continuing our investigation into the EcoRV structures responsible for the different kinetic classes of association. We have also further developed a method for stabilizing labile DNA-protein complexes for analysis by the gel mobility shift assay. The electrophoretic mobility shift assay (EMSA) is a standard and widely used tool in molecular biology for measuring DNA-protein complex formation. Many nonspecific DNA-protein complexes, however, are weak enough that they dissociate in the gel, giving smeared bands that are difficult to quantitate precisely. In order to extend the applicability of the EMSA to these labile complexes, we have investigated the effect on adding stabilizing osmolytes (such as glycerol or triethylene glycol) to the gel itself. This project is a logical continuation of our previous work on trapping DNA-protein complexes. The dissociation of complex in a gel necessarily exposes the protein and DNA surface area that was buried in the complex. The stabilizing effect of osmolytes on protein-DNA complexes can be rationalized by the exclusion of osmolytes from exposed protein and DNA surfaces. In this work, we have focused on complexes of the restriction endonuclease EcoRI with nonspecific and noncognate star sequence oligonucleotides. Our results clearly demonstrate that including the osmolyte triethylene glycol in the gel dramatically stabilizes both the weak star and nonspecific complexes of EcoRI. Without solute, both non-cognate and nonspecific complexes simply dissociate too quickly in 10% polyacrylamide gels. We showed that 30% triethylene glycol in the gel (equivalent to 4.3 osmolal) is enough to stabilize completely complexes that have dissociation constants at regular salt and pH conditions in the micromolar range. The technique can be readily used for even weaker complexes and in principle for any RNA and DNA-protein complex that is sensitive to osmotic stress. Extension of this approach to other techniques for separating complex and free components as gel chromatography and capillary electrophoresis is straightforward. Results are published in the Electrophoresis Journal.

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。我们利用我们开发的独特的实验工具来研究这些复合物的结构，热力学和功能的耦合。 我们目前正在研究II型限制酶Ecorv的DNA复合物。通常，限制性核酸内切酶可以在没有裂解所需的二价金属辅助因子的情况下，很有效地区分特定识别和非特异性DNA序列。然而，目前，文献中的结果表明，Ecorv的序列严格性异常低。对于ECORV裂解活性是最佳的pH 7.5的大多数研究，发现在没有二价离子的情况下，Ecorv结合常数与特异性和非特异性DNA序列之间的差异不到10倍。但是，文献中有一个测量与缺乏认识的严格性相矛盾。此外，在没有金属co因子的情况下解决的特定和非认知DNA-ECORV复合物的X射线晶体结构明显不同。 特定复合物的界面基本上是无水的，与许多直接DNA-蛋白相互作用相互作用，与非共同认知复合物有很大不同，该复合物在蛋白质-DNA界面上具有较大的水填充间隙，这表明水合和结合自由能之间的显着差异两个配合物。用于测量ECORV-DNA结合的生化技术主要是凝胶迁移率转移测定法和多种荧光技术，所有这些技术都容易发生伪像。 我们已经应用了以前由我们开发的自切除测定法，以测量溶液中的ECORV-DNA结合。该技术仅监测核酸内切酶的酶促复合物。它没有凝胶迁移率转移测定的局限性，同时提供了相同的灵敏度。 均衡测量尤其需要了解关联率的知识。我们发现，在没有二价离子的情况下，ECORV对特定复合物的形成具有相当不寻常的动力学，而ECORI未观察到。 总酶的很大一部分（45％）形成酶竞争的复合物异常缓慢，尤其是在pH 7.6时。这种新颖的结果可以通过溶液中游离酶的两个构象之间非常缓慢的过渡来解释。缓慢而快速关联的蛋白质结构及其交换动力学的平衡分布可能取决于许多参数，包括pH，盐，渗透源和二价阳离子。复杂形成的速度缓慢可以解释其他人报告的缺乏特异性。 我们已经使用自切除测定法测量了特定和非特异性结合常数的比率，从而提供了达到平衡所需的较长孵化时间。在pH 7.6时，与310 bp片段的结合常数比与30 bp非特异性寡核苷酸的结合常数高60倍。这大约比通常观察到的数量级大。相对特异性非特异性结合常数KNSP-SP随着pH值的降低和中性渗透浓度的增加而大大增加。相对结合常数的渗透压依赖性仅对pH弱敏感，表明特异性和非特异性复合物的结构反映在隔离水在pH下的不同变化中的差异所反映。对KNSP-SP的观察到的大渗透依赖性表示，稀释溶液中蛋白-DNA特异性的测量不能直接应用于细胞拥挤环境中的结合。除二价离子外，水活性和pH是两个关键参数，它们强烈调节ECORV的结合特异性。 至少有两个动力学成分的观察结果表明，ECORV是一种具有至少两个构象的变构蛋白。现在，变构被认为是DNA蛋白复合物的重要概念，从而提供了对结合和活性的额外控制水平。 DNA结合蛋白的识别特异性或活性可以通过与其他人相对于其他变形构象结合的配体或蛋白质调节。我们正在继续研究负责不同动力学阶层的ECORV结构。 我们还进一步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。电泳迁移率转移测定法（EMSA）是用于测量DNA-蛋白质复合物形成的标准且广泛使用的工具。然而，许多非特异性DNA蛋白复合物的弱较弱，以至于它们在凝胶中解离，从而产生了难以精确定量的涂抹带。为了将EMSA的适用性扩展到这些不稳定的复合物中，我们研究了将稳定渗透液（例如甘油或三乙二醇）添加到凝胶本身的影响。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。凝胶中复合物的解离必定会暴露于埋在复合物中的蛋白质和DNA表面积。通过排除暴露的蛋白质和DNA表面中的渗透液，可以合理地将渗透液对蛋白DNA复合物的稳定作用合理化。在这项工作中，我们专注于限制性核酸内切酶Ecori的复合物，该复合物具有非特异性和非认知星序列寡核苷酸。我们的结果清楚地表明，在凝胶中包括三乙二醇在内，可以显着稳定ECORI的弱星和非特异性复合物。在没有溶质的情况下，非认知和非特异性复合物都简单地在10％聚丙烯酰胺凝胶中分离过。我们表明，凝胶中的30％三甲基乙二醇（相当于4.3渗透）足以稳定完全在微摩尔范围内在常规盐和pH条件下具有解离常数的完全复合物。该技术可以轻松用于甚至较弱的复合物，并且原理可以用于对渗透应激敏感的任何RNA和DNA蛋白质复合物。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。结果发表在《电泳杂志》中。