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，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。我们已经应用了以前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二价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。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值的降低而大大增加。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，较大的渗透压依赖性意味着在稀释溶液中蛋白-DNA特异性的测量不能直接应用于细胞的乌鸦环境中的结合。除二价离子外，水活性和pH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。我们观察到的ECORV复合物形成的缓慢速率必须决定孵育时间的时间比典型达到平衡的时间更长，尤其是在高于7.0的pH值下。这可能解释了竞争性结合常数的某些差异。 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。 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。在关联中，蛋白质最初是非特异性地结合并沿着DNA滑动的，以找到特定的序列或分离，然后重新开始过程。滑动使蛋白质可以扫描DNA区域。它还使蛋白质能够比溶液中允许的扩散更快地定位其识别序列。如果通常会被核小体阻塞的转录因子的识别位点，则可能有一个有限的时间段内，在此期间，核小体瞬间移位以使这些因子找到其位点。分离和随后的关联步骤之间的期限称为跳跃或跳跃时间。解离过程恰恰相反；特异性结合的蛋白将过渡到识别位点的非特异性结合形式，并开始沿着DNA滑动以重新点到特定位点或将其分离为溶液。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带有预动蛋白的较长DNA片段的溶液中来测量解离动力学。从DNA片段中解离的蛋白质被添加的竞争者捕获。我们使用凝胶迁移率转移测定变体或一种自我切割测定法，我们已经开发出来测量片段结合蛋白随时间的损失。特异性位点浓度的寡核苷酸和DNA片段的比率至少为100，通常更高。在这些高比率下，如果将蛋白质添加到两者的混合物中，则蛋白质与DNA片段结合的可能性小于1％。但是，我们发现ECORI和ECORV的分离动力学都取决于寡核苷酸的浓度。我们推测，这种依赖性是由于蛋白跳动动力学所致。在蛋白质与DNA的初始解离之后，它仍然接近DNA。蛋白质重新启动到同一DNA的可能性很大。数学表达式可用于分布时间0的蛋白质在时间t上分离到原始DNA片段的时间，假设没有其他DNA在时间t。在此暂停期间，蛋白质可以结合寡核苷酸。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蛋白质在跳跃过程中被寡核苷酸捕获的概率。由随机行走模拟确定的分布函数对观察到的ECORI观察到的寡核苷酸浓度依赖性提供了相当好的描述。但是，二维第一通道时间的分析表达预测的依赖性要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，解离速率对ECORV的寡核苷酸浓度的依赖性对pH敏感。在低pH值下，寡核苷酸浓度依赖性与ECORI大致相同。 pH 7.5的依赖性少得多。我们的工作假设是，返回DNA并不一定意味着回到识别地点。在再次到达识别位点之前，蛋白质会再次解离。在没有重现特定序列的概率的极限下，将没有寡核苷酸浓度依赖性。该pH可以影响这种概率意味着pH会影响相对滑动和解离速率，或者蛋白质可以在pH依赖性构象中解离，而pH依赖性构象无法重新启动到DNA。后一种可能性很有吸引力，因为搜索过程将更有效。蛋白质必须从原始结合区域进一步扩散，然后才能重现为DNA。 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。我们已经表明，凝胶中的30％三乙二醇（相当于4.3渗透）足以稳定完全弱的复合物，这些复合物在微摩尔范围内的常规盐和pH条件下具有解离常数。现在，我们将这种方法进一步扩展到其他技术，以将复合物和游离成分分开为凝胶色谱和毛细管电泳。