Recognition reactions between macromolecules

大分子之间的识别反应

• 批准号: 8351203
• 负责人: Donald Rau
• 金额: $ 25.22万
• 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/8351203
• 关键词: Accounting; Affect; Binding; Binding Proteins; Biological Assay; Biological Models; Capillary Electrophoresis; Cell physiology; Cells; Competitive Binding; Complex; Coupling; DNA; DNA Restriction Enzymes; DNA Sequence; DNA-Binding Proteins; Dependence; Diffuse; Diffusion; Dissociation; Divalent Cations; Electrostatics; Environment; Enzymes; Equilibrium; Explosion; Gel; Gel Chromatography; Goals; Hop protein; Humulus; Hydration status; Individual; Investigation; Ions; Kinetics; Laboratories; Ligands; Literature; Macromolecular Complexes; Measurement; Measures; Methods; Molecular Conformation; Monitor; Nucleic Acids; Nucleosomes; Oligonucleotides; Osmotic Pressure; Play; Probability; Process; Protein Binding; Proteins; Reaction; Relative (related person); Reporting; Role; Scanning; Sequence-Specific DNA Binding Protein; Site; Slide; Sodium Chloride; Solutions; Specificity; Stress; Structure; Techniques; Thermodynamics; Time; Type II site-specific deoxyribonuclease; Variant; Walking; Water; Work; divalent metal; endonuclease; gel mobility shift assay; macromolecule; novel; preference; protein complex; protein structure; simulation; solute; tool; transcription factor; triethylene glycol; two-dimensional

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. We have applied a self-cleavage assay, developed previously 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. 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 means that measurement of protein-DNA specificity in dilute solution cannot be directly applied to binding in the crowed environment of the cell. In addition to divalent ions, water activity and pH are key parameters that strongly modulate binding specificity of EcoRV. 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. The slow rate of EcoRV complex formation we observe necessarily dictates longer incubation times than is typical to reach equilibrium especially at pH values higher than 7.0. This may account for some of the difference in competitive binding constants. 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. The association and dissociation kinetics of sequence specific DNA binding proteins are surprisingly complicated. In association, proteins initially bind nonspecifically and slide along the DNA to either find the specific sequence or dissociate and start the process again. Sliding allows the protein to scan a region of DNA. It also enables the protein to locate its recognition sequence faster than diffusion in solution would allow. If the recognition sites of transcription factors are normally occluded by nucleosomes, there may be a limited time period during which nucleosomes are transiently displaced for these factors to find their sites. The period between dissociation and subsequent association steps is termed the hopping or jumping time. The dissociation process is just the opposite; the specifically bound protein will transition to a nonspecifically bound form at the recognition site and start sliding along the DNA to either rebind to the specific site or dissociate into solution. We have uncovered a novel method to probe the hopping process. Dissociation kinetics can be measured by adding oligonucleotide containing the specific site to a solution of a longer DNA fragment with prebound protein. Protein that dissociates from the DNA fragment is trapped by the added competitor. We use a gel mobility shift assay variant or a self-cleavage assay that we have developed to measure the loss of fragment bound protein with time. The ratio of specific site concentrations of oligonucleotide and of DNA fragment is at least 100 and is usually higher. At these high ratios, if protein was added to the mixture of the two, the probability that the protein will bind to the DNA fragment is <1%. We find, however, that the dissociation kinetics of both EcoRI and EcoRV depend on the oligonucleotide concentration. We surmise that this dependence is due to protein hopping kinetics. After the initial dissociation of protein from DNA, it is still close to the DNA. The probability that the protein will rebind to the same DNA is quite large. Mathematical expressions are available for the distribution of times that a protein that dissociates at time 0 will rebind to the original DNA fragment at time t assuming no other DNA is around. During this off-time the protein can bind the oligonucleotide. The probability that a protein will be captured by an oligonucleotide during a hopping excursion can be calculated from the oligonucleotide concentration, the association rate constant, and the hopping time distribution function. A distribution function determined by random walk simulations gives a reasonably good description of the oligonucleotide concentration dependence observed for EcoRI. The analytical expression for 2-dimensional first passage times, however, predicts a much smaller dependence than is observed. We suspect that the electrostatic attraction between protein and DNA is the reason the analytic expression fails. Unlike EcoRI, the dependence of dissociation rate on oligonucleotide concentration for EcoRV is pH sensitive. At low pH values the oligonucleotide concentration dependence is about the same as for EcoRI. There is much less dependence at pH 7.5. Our working hypothesis is that a return to the DNA does not necessarily mean a return to the recognition site; there is a probability that the protein will dissociate again before reaching the recognition site again. In the limit of no probability of rebinding of the specific sequence there will be no oligonucleotide concentration dependence. That pH can affect this probability means that either pH affects the relative sliding and dissociation rates or that the protein can dissociate in a pH dependent conformation that is not able to rebind to the DNA. This latter possibility is attractive since the search process will be more efficient; the protein must diffuse further from the original binding region before being able to rebind to DNA. We have also further developed a method for stabilizing labile DNA-protein complexes for analysis by the gel mobility shift assay. We have shown that 30% triethylene glycol in the gel (equivalent to 4.3 osmolal) is enough to stabilize completely weak complexes that have dissociation constants at regular salt and pH conditions in the micromolar range. We are now further extending this approach to other techniques for separating complex and free components as gel chromatography and capillary electrophoresis.

我们目前正在研究 II 型限制酶 EcoRV 的 DNA 复合物。通常，在缺乏切割所需的二价金属辅助因子的情况下，限制性内切核酸酶可以非常有效地区分特异性识别和非特异性 DNA 序列。然而，目前文献结果表明 EcoRV 的序列严格性异常低。我们应用了我们之前开发的自裂解测定法来测量 EcoRV-DNA 竞争性结合，并评估在不存在二价离子的情况下水活度、pH 和盐浓度对酶结合严格性的影响。该技术仅监测核酸内切酶的酶活性复合物。它没有凝胶迁移率变化测定的局限性，同时提供相同水平的灵敏度。我们发现该酶可以很容易地区分特异性和非特异性序列。相对特异性-非特异性结合常数随着中性溶质浓度的增加和 pH 值的降低而强烈增加。特异性和非特异性DNA-EcoRV复合物之间缔合水数量的差异与晶体结构的差异一致。尽管序列特异性对 pH 值有很大依赖性，但渗透压依赖性表明结构随 pH 值变化很小。重要的是，大的渗透压依赖性意味着稀溶液中蛋白质-DNA特异性的测量不能直接应用于细胞拥挤环境中的结合。除了二价离子外，水活度和 pH 值也是强烈调节 EcoRV 结合特异性的关键参数。 我们发现，在没有二价离子存在的情况下，EcoRV 具有非常不寻常的特定复合物形成动力学，而这一点在 EcoRI 中未观察到。总酶的很大一部分（45%）形成酶活性复合物的速度异常缓慢，特别是在 pH 7.6 时。这一新颖的结果可以通过溶液中游离酶的两种构象之间非常缓慢的转变来解释。缓慢和快速缔合蛋白质结构的平衡分布及其交换动力学可能取决于许多参数，包括 pH、盐、渗透剂和二价阳离子。复合物形成速度缓慢可以解释其他人报道的缺乏特异性的原因。我们观察到的 EcoRV 复合物形成速度较慢，必然需要比通常达到平衡更长的孵育时间，尤其是在 pH 值高于 7.0 的情况下。这可能解释了竞争性结合常数的一些差异。 对至少两个相关动力学成分的观察表明EcoRV是具有至少两种构象的变构蛋白。变构现象现在被认为是 DNA-蛋白质复合物的重要概念，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优先与一种变构构象结合的配体或蛋白质来调节。我们正在继续研究负责不同动力学类别关联的 EcoRV 结构。 序列特异性 DNA 结合蛋白的缔合和解离动力学异常复杂。在关联中，蛋白质最初非特异性结合并沿着 DNA 滑动以找到特定序列或解离并再次开始该过程。滑动使蛋白质能够扫描 DNA 区域。它还使蛋白质能够比溶液中扩散更快地定位其识别序列。如果转录因子的识别位点通常被核小体封闭，则可能存在一段有限的时间段，在此期间核小体暂时移位，以便这些因子找到它们的位点。解离和随后的缔合步骤之间的时期称为跳跃或跳跃时间。解离过程正好相反；特异性结合的蛋白质将在识别位点转变为非特异性结合形式，并开始沿着 DNA 滑动，重新结合到特定位点或解离到溶液中。我们发现了一种探测跳跃过程的新方法。 可以通过将含有特定位点的寡核苷酸添加到具有预结合蛋白质的较长 DNA 片段的溶液中来测量解离动力学。从 DNA 片段解离的蛋白质被添加的竞争物捕获。我们使用我们开发的凝胶迁移率变化测定法或自切割测定法来测量片段结合蛋白随时间的损失。寡核苷酸和DNA片段的特定位点浓度的比率至少为100并且通常更高。在如此高的比例下，如果将蛋白质添加到两者的混合物中，则蛋白质与 DNA 片段结合的概率 <1%。然而，我们发现 EcoRI 和 EcoRV 的解离动力学都取决于寡核苷酸浓度。我们推测这种依赖性是由于蛋白质跳跃动力学造成的。蛋白质与DNA初步解离后，仍然与DNA很接近。蛋白质与相同 DNA 重新结合的可能性相当大。数学表达式可用于表示在时间 0 解离的蛋白质将在时间 t 重新结合到原始 DNA 片段（假设周围没有其他 DNA）的时间分布。在此期间，蛋白质可以结合寡核苷酸。蛋白质在跳跃偏移期间被寡核苷酸捕获的概率可以根据寡核苷酸浓度、缔合速率常数和跳跃时间分布函数来计算。由随机游走模拟确定的分布函数给出了对 EcoRI 观察到的寡核苷酸浓度依赖性的相当好的描述。然而，二维首次通过时间的解析表达式预测的依赖性比观察到的要小得多。我们怀疑蛋白质和 DNA 之间的静电吸引力是分析表达失败的原因。与 EcoRI 不同，EcoRV 的解离速率对寡核苷酸浓度的依赖性对 pH 值敏感。在低 pH 值下，寡核苷酸浓度依赖性与 EcoRI 大致相同。 pH 值为 7.5 时依赖性要小得多。我们的工作假设是，DNA 的回归并不一定意味着识别位点的回归；相反，DNA 的回归并不一定意味着识别位点的回归。蛋白质在再次到达识别位点之前有可能再次解离。在特定序列不可能重新结合的限度内，将不存在寡核苷酸浓度依赖性。 pH 值会影响这种概率，这意味着 pH 值会影响相对滑动和解离速率，或者蛋白质会以 pH 依赖性构象解离，而无法与 DNA 重新结合。后一种可能性很有吸引力，因为搜索过程将更加高效；在能够与 DNA 重新结合之前，蛋白质必须从原始结合区域进一步扩散。 我们还进一步开发了一种稳定不稳定 DNA-蛋白质复合物的方法，用于通过凝胶迁移率变动分析进行分析。我们已经证明，凝胶中 30% 三甘醇（相当于 4.3 渗透压）足以稳定完全弱的复合物，这些复合物在常规盐和 pH 条件下的解离常数在微摩尔范围内。我们现在将这种方法进一步扩展到其他分离复杂和游离成分的技术，如凝胶色谱和毛细管电泳。