Recognition reactions between macromolecules

大分子之间的识别反应

• 批准号: 8553934
• 负责人: Donald Rau
• 金额: $ 32.37万
• 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/8553934
• 关键词: Bacteria; Base Pairing; Binding; Biological; Biological Assay; Biological Models; Cell physiology; Cells; Charge; Cleaved cell; Competitive Binding; Complex; Coupling; DNA; DNA Binding; DNA Restriction Enzymes; DNA Sequence; Deoxyribonuclease EcoRI; Dependence; Diffuse; Diffusion; Dissociation; Enterobacteriaceae; Environment; Enzymes; Equilibrium; Exhibits; Explosion; Genes; Goals; Humulus; Hydration status; Individual; Investigation; Ions; Kinetics; Laboratories; Literature; Macromolecular Complexes; Measurement; Measures; Methods; Modeling; Molecular; Molecular Chaperones; Molecular Conformation; Monitor; Nucleic Acids; Oligonucleotides; Osmotic Pressure; Phase; Physiological; Play; Probability; Protein Binding; Proteins; Reaction; Relative (related person); Reporting; Role; Sequence-Specific DNA Binding Protein; Sodium Chloride; Solutions; Specificity; Stomach; Stress; Structure; System; Techniques; Thermodynamics; Time; Type II site-specific deoxyribonuclease; Water; endonuclease; gel mobility shift assay; macromolecule; novel; protein complex; protein structure; restriction enzyme; solute; tool; uptake

We are continuing to investigate DNA complexes of the type II restriction enzyme, EcoRV. DNA binding stringency is crucial for proper function of restriction enzymes . At present, however, results in literature suggest that EcoRV has unusually low sequence stringency in the absence of divalent ions. We have applied a self-cleavage assay, developed by us, to measure EcoRV-DNA competitive binding and to evaluate the influence of water activity, pH and salt concentration on the binding stringency of the enzyme in the absence of divalent ions. 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. We find the enzyme can readily distinguish specific and nonspecific sequences. The relative specific-nonspecific binding constant increases strongly with increasing neutral solute concentration and with decreasing pH indicating that water activity and pH are key parameters that strongly modulate binding specificity of EcoRV in addition to divalent ions. The difference in number of associated waters between specific and nonspecific DNA-EcoRV complexes is consistent with the differences in the crystal structures. Despite the large pH dependence of the sequence specificity, the osmotic pressure dependence indicates little change in structure with pH. Importantly, the large osmotic pressure dependence we saw for the EcoRV and EcoRI restriction endonucleases as well as for gal and cro bacterial repressors means that measurement of protein-DNA specificity in dilute solution cannot be directly applied to binding in the crowed environment of the cell. The time needed to reach equilibrium depends on association and dissociation rates. 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 slow transition between two conformations (opened or closed) of the free enzyme in solution. We found that both specific and nonspecific DNA shifts equilibrium distribution toward opened or fast binding form of the protein playing a role of the allosteric regulator or a chaperone. We are continuing our investigation into the EcoRV structures responsible for the different kinetic classes of association. Restriction endonucleases major biological role is to protect bacteria from foreign DNA invasion. In the presence of Mg2+ ions restriction endonucleases become extremely precise molecular scissors cleaving foreign (unmethylated) DNA with exquisite specificity. The optimal enzymatic activity of EcoRV is reached at about pH 7.5. Cleavage activity decreases dramatically with decreasing pH while specific equilibrium binding and binding specificity strongly increase. We are now exploring the influence of pH and osmotic stress on Mg2+ binding to the EcoRV-DNA complex using self-cleavage assay. This technique allows us to measure Mg2+ binding on an exceptionally slow time-scale. We find that the time dependence of the fraction DNA cleaved after incubation of EcoRV-DNA complexes with Mg2+ exhibits a lag phase and cannot be fit with single exponential. The kinetics can be well fit with two exponential functions. The binding of Mg2+ to the pre-formed EcoRV-DNA complex involves at least two steps and DNA can only be cleaved after the second step. Each binding step is characterized by an uptake of about 30-40 water molecules, the binding of 1-2 Mg2+ ions, and the release of 1-2 H+ ions. We hypothesize that the two steps correspond to the separate binding of Mg2+ to the two enzyme subunits. The interplay of Mg2+ and pH might play a very important role in the survival mechanism of enteric bacteria that spends significant amount of time at very low pH conditions of the stomach. The association and dissociation kinetics of sequence specific DNA binding proteins are surprisingly complicated. It is generally thought that the sequence specific DNA binding proteins that regulate gene activity locate their target sequences by initially binding nonspecifically with subsequent one-dimensional diffusion along DNA interspersed with short hops or jumps and by direct transfer of protein from one DNA helix to another. Direct transfer is characterized by the formation of an intermediate DNA-protein-DNA ternary complex. Consequently, the dissociation rate depends on total DNA concentration. The direct transfer model is gaining popularity since protein-DNA dissociation rates are becoming more widely observed to depend on competitor DNA concentrations. Since most all specific or nonspecific complexes of these proteins with DNA entail charge-charge interactions, the salt concentration dependence of dissociation due to direct transfer and formation of a ternary DNA-protein complex should be quite different than from simple dissociation. As the salt concentration is increased, the contribution to dissociation from direct transfer should significantly decrease. This difference, however, has not been directly investigated. We have found that at near physiological NaCl concentrations the dissociation of the restriction endonuclease EcoRI from specific sequence DNA has both competitor DNA concentration dependent and independent contributions. At two-fold higher salt concentrations, only the competitor oligonucleotide concentration independent component is observed as expected for smaller salt concentration dependence of direct transfer compared to simple dissociation. Unexpectedly, however, no difference is seen in the salt concentration dependence of dissociation measured around physiological salt concentration for two concentrations of competitor oligonucleotide that show quite different apparent contributions from direct transfer. These disparate observations can be rationalized by considering the physical consequences of transient protein dissociation. After a protein dissociates from DNA fragment, for a short period of time it remains very close and has high probability to simply rebind to the same DNA. The probability that the protein will rebind after a time t is given by the first passage time distribution function that has been estimated for DNA-protein systems. During the time the protein is off the DNA, there is also a probability it will react and bind with competitor DNA. This probability will, of course, depend on competitor DNA concentration and the association rate. A calculation of the probability of reaction with a competitor oligonucleotide before the protein rebinds to the initial DNA indicates that this mechanism does fit the experimentally observed dependence of the dissociation rate on competitor concentration at physiological salt concentrations. Since the protein has dissociated from the initial DNA and no ternary complex is formed, the salt dependence will be independent of oligonucleotide concentration. The loss of oligonucleotide concentration dependence with doubling the salt concentration can also be understood within this mechanism. At physiological salt concentrations, we were able to determine previously that the protein can diffuse hundreds of base pairs along the DNA, far enough such that after rebinding the protein has a high probability to find the specific recognition sequence before dissociating again. At twice the salt concentration, however, the protein can only diffuse short distances along the DNA (tens of base pairs instead of hundreds). It is highly unlikely that the protein can find the specific sequence before dissociating again. Reaction with the competitor DNA is much more probable now at all practical oligonucleotide concentratio

我们正在继续研究II型限制酶Ecorv的DNA复合物。 DNA结合严格度对于限制酶的正确功能至关重要。然而，目前，文献中的结果表明，在没有二价离子的情况下，ECORV具有异常低的序列严格度。我们已经应用了由我们开发的自切解测定法，以测量ECORV-DNA竞争性结合，并评估水活性，pH和盐浓度对酶在缺乏分离离子的情况下的结合严格度的影响。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。我们发现酶可以很容易地区分特定和非特异性序列。随着中性溶质浓度的增加，相对特异性非特异性结合常数强烈增加，并且pH值降低表明水活性和pH是关键参数，除二价离子以外，其强烈调节ECORV的结合特异性。特异性和非特异性DNA-ECORV复合物之间相关水数的数量与晶体结构的差异一致。尽管序列特异性的pH依赖性很大，但渗透压依赖性表明结构的变化很小。重要的是，我们对ECORV和ECORI限制性核酸内切酶以及GAL和CRO细菌抑制剂的巨大渗透压依赖性表示，稀释溶液中蛋白-DNA特异性的测量不能直接应用于在细胞拥挤环境中的结合。 达到平衡所需的时间取决于关联和解离率。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。这个新颖的结果可以通过溶液中游离酶的两个构象（打开或封闭）之间的缓慢过渡来解释。我们发现，特异性和非特异性DNA都将平衡分布转移到蛋白质的打开或快速结合形式中，起着变构调节剂或伴侣的作用。我们正在继续研究负责不同动力学阶层的ECORV结构。 限制性核酸内切核的主要生物学作用是保护细菌免受外源DNA侵袭。在存在Mg2+离子限制的情况下，核叶酶成为极其精确的分子剪刀，它们具有精致的特异性，它们裂解异物（未甲基化的）DNA。 ECORV的最佳酶活性在约pH 7.5处达到。裂解活性随着pH的降低而大大降低，而特异性平衡结合和结合特异性则强烈增加。现在，我们正在使用自切解测定法探索pH和渗透应激对MG2+结合与Ecorv-DNA复合物的影响。这种技术使我们能够在极慢的时间尺度上测量MG2+结合。我们发现，与Mg2+孵育ECORV-DNA复合物后，分数DNA的时间依赖性表现出滞后相，并且不能与单个指数相吻合。动力学可以很好地适合两个指数函数。 Mg2+与预成型的ECORV-DNA复合物的结合涉及至少两个步骤，并且DNA只能在第二步之后切割。每个结合步骤的特征是摄取约30-40个水分子，1-2 mg2+离子的结合和1-2 H+离子的释放。我们假设这两个步骤对应于Mg2+与两个酶亚基的单独结合。 MG2+和pH的相互作用可能在肠细菌的生存机理中起着非常重要的作用，肠细菌在胃的pH pH值中花费大量时间。 序列特异性DNA结合蛋白的缔合和解离动力学令人惊讶地复杂。人们普遍认为，调节基因活性的序列特异性DNA结合蛋白通过最初与随后的一维扩散沿DNA散布在短啤酒花或跳跃中，并直接将蛋白质从一个DNA螺旋直接转移到另一个DNA。直接转移的特征在于形成中间DNA-蛋白-DNA三元复合物。因此，解离率取决于总DNA浓度。由于蛋白质-DNA解离速率越来越广泛地观察到取决于竞争者的DNA浓度，因此直接转移模型正在越来越受欢迎。由于这些蛋白质的大多数特定或非特异性复合物都具有DNA需要电荷 - 电荷相互作用，因此由于直接转移和形成三元DNA-蛋白质复合物引起的分离盐浓度依赖性应与简单解离的盐浓度依赖性应大不相同。随着盐浓度的增加，与直接转移解离的贡献应显着降低。但是，这种差异尚未直接研究。 我们发现，在接近生理的NaCl浓度下，限制内切核酸内切酶ECORI与特定序列DNA的解离具有竞争者DNA浓度依赖性和独立贡献。与简单解离相比，在盐浓度较高的两倍时，仅观察到寡核苷酸浓度独立的成分，而直接转移的盐浓度依赖性较小。然而，出乎意料的是，在两种浓度的竞争者寡核苷酸的盐浓度依赖性的盐浓度依赖性中没有看到差异，这表明与直接转移相比显示出明显的明显贡献。 这些不同的观察结果可以通过考虑瞬时蛋白质解离的物理后果来合理化。在蛋白质与DNA片段分离后，在短时间内，它保持非常接近，并且很有可能仅重新点燃相同的DNA。蛋白质在时间t之后通过第一个通过DNA-蛋白质系统估计的时间T给出了蛋白质的概率。在蛋白质脱离DNA的过程中，它也有一种概率与竞争者DNA反应并结合。当然，这种概率将取决于竞争者DNA浓度和关联率。在蛋白质重新介绍初始DNA之前，与竞争者寡核苷酸反应的概率计算表明，这种机制确实符合实验观察到的解离率对生理盐浓度下竞争者浓度的依赖性。由于蛋白质与初始DNA分离并且没有形成三元复合物，因此盐依赖性将独立于寡核苷酸浓度。在这种机制中，寡核苷酸浓度依赖性的损失也可以理解。在生理盐浓度下，我们以前能够确定蛋白质可以沿DNA扩散数百个碱基对，以至于重新固定后，蛋白质具有很高的可能性，可以在再次解离之前找到特定的识别顺序。然而，在盐浓度的两倍处，蛋白质只能沿DNA（数十个碱基对而不是数百个）扩散短距离。在再次解离之前，蛋白质极不可能找到特定序列。现在，与竞争者DNA的反应在所有实用的寡核苷酸浓缩液中现在更有可能