Recognition reactions between macromolecules

大分子之间的识别反应

基本信息

批准号：
7968728
负责人：
Donald Rau
金额：
$ 69.25万
依托单位：
EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
依托单位国家：
美国
项目类别：
财政年份：
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/7968728
关键词：
Area Betaine Binding Binding Proteins Biological Assay Biological Models Capillary Electrophoresis Cell physiology Cells Competitive Binding Complex Coupling Crowding DNA DNA Binding DNA Restriction Enzymes DNA Sequence DNA-Protein Interaction Deoxyribonuclease EcoRI Dependence Dissociation Electrophoretic Mobility Shift Assay Ensure Environment Enzymes Equilibrium Exclusion Explosion Free Energy Gel Gel Chromatography Glycerol Goals Half-Life Hour Hydration status Individual Ions Kinetics Laboratories Life Literature Manuscripts Measurement Measures Methods Molecular Biology Nature Nucleic Acids Oligonucleotides Osmotic Pressure Play Preparation Protein Binding Proteins RNA Reaction Relative (related person)Reporting Roentgen Rays Role Sequence-Specific DNA Binding Protein Sodium Chloride Solutions Specificity Stress Structure Surface Techniques Thermodynamics Time Type II site-specific deoxyribonuclease Variant Water Work divalent metal gel mobility shift assay macromolecule novel nuclease polyacrylamide gels protein complex protein structure restriction enzyme solute tool triethylene glycol

项目摘要

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. At present, however, results in literature suggest that EcoRV has a quite low sequence stringency. 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 under the same experimental conditions. It has been suggested that EcoRV represents a new paradigm for restriction nuclease recognition. X-ray crystal structures are available for both cognate and non-cognate EcoRV complexes in the absence and in the presence of divalent ions. Because the two structures are so substantially different, it is contra intuitive that specific and nonspecific binding should barely differ in binding energy in the absence of divalent ions. 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 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. We used the self-cleavage assay to quantitate EcoRV-DNA binding. Kinetic studies are necessary to ensure that equilibrium studies can be performed properly. We found that EcoRV association kinetics is very complicated. 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 that can range from about 20 min. at pH 6.9 up to an hour at pH 7.6. This is far different from our previous observations with EcoRI. The slow component of the association kinetics indicates that at least 2 hours incubation pH 6.9 and even longer time at pH 7.6 is necessary to reach equilibrium. We consider the unusual association kinetics of EcoRV the most promising of our findings for explaining the significant variation in the specific equilibrium constant values reported by different groups. 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 found that EcoRV can effectively distinguish between cognate and nonspecific DNA sequences in the absence of divalent co-factors though, as was shown before, divalent co-factors dramatically increase EcoRV binding selectivity. We demonstrated that both pH and osmotic stress are also critically important for the ability of the enzyme to bind DNA in a specific manner. Ratio between specific and nonspecific binding constants increases from 56 at pH 7.6, to 280 at pH 6.9, and to 1085 at pH 6.2 in the absence of osmolytes. Even at pH 7.6, the ratio between specific and nonspecific binding constants increases from 56 in the absence of neutral solutes to 3300 in the presence of 1 osmolal triethylene glycol, mimicking the crowded environment of the living cell. EcoRV should not be considered a representative of a different class of restriction enzymes that is capable of sequence recognition at the cleavage step of the reaction only. We found that the free energy difference between specific and nonspecific EcoRV complexes is linearly dependent on solute osmolal concentrations for each of the four solutes used. 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. We found that the difference in hydration between specific and nonspecific EcoRV complexes depends on solute nature changing from 116 water molecules measured for betaine up to 240 water molecules measured for the triethylene glycol. This result suggests significant difference in surface exposed area between two complexes. Manuscript is in preparation for this part of the project. 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 star sequence oligonucleotide. 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.

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，限制内切核酸酶可以在没有二价金属co因子的情况下非常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的序列严格性很低。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。已经提出，ECORV代表了限制核酸酶识别的新范式。 X射线晶体结构在不存在和存在二价离子的情况下可用于同源和非共同的Ecorv复合物。由于这两个结构是如此之大，因此直观的是，在没有二价离子的情况下，特异性和非特异性结合在结合能上几乎没有差异。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。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。我们使用自切割测定法来定量ECORV-DNA结合。要确保可以正确进行平衡研究，动力学研究是必要的。我们发现ECORV协会动力学非常复杂。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，半衰期的时间约为20分钟。在pH 7.6时，pH值为6.9，最多一个小时。这与我们以前对ECORI的观察有很大不同。关联动力学的缓慢成分表明，至少需要2小时的pH 6.9，甚至在pH 7.6时甚至更长的时间才能达到平衡。我们认为ECORV的异常关联动力学是我们发现的最有希望的，用于解释不同组报告的特定平衡恒定值的显着差异。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）。我们发现，ECORV可以有效地区分同源和非特异性DNA序列，但是在没有二价辅助因子的情况下，如前所述，二价co骨因子显着提高了Ecorv结合的选择性。我们证明，pH和渗透应力对于以特定方式结合DNA的能力也至关重要。特异性和非特异性结合常数之间的比率从pH 7.6时的56增加到pH 6.9时的280，而在没有渗透液的情况下，在pH 6.2时为1085。即使在pH 7.6时，在存在1个渗透型三甲基乙二醇的情况下，特异性和非特异性结合常数之间的比率也从没有中性溶质的56增加到3300，模仿了活细胞拥挤的环境。不应将ECORV视为代表不同类别的限制酶，该酶仅在反应的裂解步骤下才能序列识别。我们发现，特异性和非特异性Ecorv复合物之间的自由能差是线性取决于所用溶质中每种溶质的溶质渗透浓度。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。我们发现，特异性和非特异性ECORV复合物之间的水合差异取决于溶质性质，其溶质性质是从对三乙二醇测得的240个水分子测得的116个水分子变化。该结果表明两个复合物之间的表面暴露面积有显着差异。手稿正在为项目的这一部分做准备。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，我们专注于与非特异性和星序寡核苷酸的限制性内核酸酶Ecori的复合物。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。