Assembly Forces And Recognition Reactions

装配力和识别反应

• 批准号: 7968653
• 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
• 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/7968653
• 关键词: Arginine; Bacteria; Binding; Biological; Biopolymers; Carbohydrates; Cell Nucleus; Cell physiology; Characteristics; Charge; Chemicals; Complex; Coupling; DNA; DNA Damage; DNA Packaging; DNA Repair; DNA Sequence; Disease; Dissociation; Electrostatics; Entropy; Equilibrium; Exclusion; Free Energy; Free Radicals; Goals; Group Structure; Hydration status; Image; In Vitro; Infertility; Ions; Knowledge; Length; Link; Lipid Bilayers; Literature; Magnetism; Male Infertility; Measurement; Measures; Nature; Neutral Amino Acids; Osmotic Pressure; Pentas; Peptides; Physics; Protamines; Protein Binding; Proteins; Reaction; Reactive Oxygen Species; Reporting; Residual state; Role; Salmon; Salts; Sequence-Specific DNA Binding Protein; Solutions; Solvents; Specificity; Sperm Head; Stress; Structure; Surface; System; Testing; Touch sensation; Vertebral column; Virus; Water; aqueous; base; design; human male; in vivo; inorganic phosphate; macromolecule; male; nanometer; nucleic acid structure; planetary Atmosphere; polyarginine; reconstitution; research study; single molecule; solute; sperm cell; structural biology; theories

Dense packaging DNA in eukaryotic nuclei, sperm nuclei, viruses, and bacteria is necessary for proper cellular functioning. DNA assembly by multivalent ions is a critical testing ground for understanding not only in vivo compaction of DNA but also the physics of interactions between charged molecules. If a sufficient concentration of multivalent ions is present, DNA will spontaneously assemble into an ordered array. The helices do not collapse to touching but are rather separated by 0.5-1.5 nm of solvent depending on the nature of the condensing ion. Attractive and repulsive forces balance at the equilibrium spacing. By combining the osmotic stress, pushing experiments with single molecule, magnetic tweezers, pulling experiments, we were able to separate the attractive and repulsive free energies at the equilibrium spacing for the several commonly used condensing agents. The results confirmed our previous hypotheses for hydration forces. There is a 0.25 nm decay length exponential repulsive force that is the hydration equivalent of the image charge repulsive force in electrostatics. The hydration atmosphere extending from a solvated surface stabilizes water structuring at the surface. Disruption of the atmosphere by replacing water with another surface will lower hydration energies regardless of the water structuring on the other surface. Repulsion should depend predominately on the water structuring of groups on the DNA surface and perhaps on the mode of binding (phosphate backbone or grooves), but not on the correlations of these groups with apposing helices. The attraction is also characterized by an exponential force but with twice the decay length (0.5 nm) of the repulsive force. Attraction results from the direct interaction of surface hydration structures. Perturbations in water structure around one surface due to the close presence of another surface can either weaken or strengthen hydration energies depending on the mutual structuring water. We postulated that the attractive force had the same exponential 0.5 nm decay length as observed previously for repulsive hydration forces, but that the force was now attractive because of correlations in complementary water structuring on apposing helices. By further investigating biogenic alkylamines and arginine peptides of varying lengths, we determined that the magnitude of the attractive force increases with the number of charges consistent with a constant loss in entropy from correlating a single molecule regardless of charge, but a gain in interaction energy that increases with the number of charges. The repulsive force is only slightly dependent on the number of charges, as expected, but does depend on the chemical nature of the bound counterion. We are now investigating packaging forces of salmon protamine assembled DNA. Protamines are small, arginine-rich peptides used to package DNA in sperm heads. Protamine-DNA forces resemble polyarginine-DNA interactions. The equilibrium spacing between DNA helices with protamine, however, corresponds to tetra- or penta-arginine, not the 21 arginine charges actually present. We have separated protamine DNA forces into their attractive and repulsive components. The attractive force magnitude itself is very close to that expected for 21 arginine charges based on our previous measurements of arginine peptides. The repulsive force, however, is significantly larger than expected from the arginine peptide measurements, suggesting that the neutral amino acids present in protamine result in an increased residual repulsion. We have confirmed this hypothesis by measuring DNA force curves with synthetic hexa-arginines that incorporate increasing numbers of neutral amino acids We have also measured DNA assembly forces in salmon sperm nuclei. The in vivo sperm and in vitro, reconstituted DNA-salmon protamine forces are virtually identical. These force measurements thus have direct biological application. We can use our knowledge of DNA assembly forces to investigate the connection between DNA packaging in sperm heads, DNA damage, and male infertility. There are several reports in the literature correlating human male infertility with protamine abnormalities. Cumulative DNA damage due to reactive oxygen species is a major direct contributor to male infertility since DNA repair systems are absent in sperm. We hypothesize that incorrect assembly of DNA by insufficient or altered protamines will result in less tightly packaged DNA in sperm (a larger than normal spacing between helices) and, consequently, a greater potential exposure of DNA to oxidizing free radicals. We can test this hypothesis by direct measurements of the distance between helices in sperm of normal and infertile males and of the accumulated DNA damage.

真核细胞核、精子核、病毒和细菌中密集包装的 DNA 对于细胞的正常功能是必要的。多价离子组装 DNA 是了解体内 DNA 压缩以及带电分子之间相互作用的物理原理的重要试验场。如果存在足够浓度的多价离子，DNA 将自发组装成有序阵列。螺旋不会塌陷到接触，而是被 0.5-1.5 nm 的溶剂分开，具体取决于凝聚离子的性质。吸引力和排斥力在平衡间距处平衡。通过结合渗透应力、单分子推动实验、磁力镊子、牵引实验，我们能够分离几种常用凝聚剂在平衡间距下的吸引和排斥自由能。结果证实了我们之前关于水合力的假设。存在一个 0.25 nm 衰减长度指数排斥力，它是静电学中图像电荷排斥力的水合当量。从溶剂化表面延伸的水合气氛稳定了表面的水结构。通过用另一个表面替换水来破坏大气将降低水合能，而不管另一个表面上的水结构如何。排斥力应主要取决于 DNA 表面基团的水结构，或许还取决于结合模式（磷酸盐主链或凹槽），但不取决于这些基团与并列螺旋的相关性。吸引力的特征还在于指数力，但衰减长度是排斥力的两倍（0.5 nm）。吸引力是由表面水化结构的直接相互作用产生的。由于另一个表面的紧密存在而导致一个表面周围的水结构的扰动可能会减弱或增强水合能，具体取决于水的相互结构。我们假设吸引力具有与先前观察到的排斥水合力相同的指数 0.5 nm 衰减长度，但由于并列螺旋上互补水结构的相关性，该力现在具有吸引力。通过进一步研究不同长度的生物烷基胺和精氨酸肽，我们确定吸引力的大小随着电荷数量的增加而增加，这与无论电荷如何关联单个分子所导致的熵的恒定损失一致，但相互作用能的增益随着收费数量的增加而增加。正如预期的那样，排斥力仅略微依赖于电荷数量，但确实依赖于结合的抗衡离子的化学性质。 我们现在正在研究鲑鱼鱼精蛋白组装 DNA 的包装力。鱼精蛋白是富含精氨酸的小肽，用于将 DNA 包装在精子头部中。鱼精蛋白-DNA 力类似于聚精氨酸-DNA 相互作用。然而，DNA 螺旋与鱼精蛋白之间的平衡间距对应于四精氨酸或五精氨酸，而不是实际存在的 21 个精氨酸电荷。我们已将鱼精蛋白 DNA 力分为吸引力成分和排斥成分。根据我们之前对精氨酸肽的测量，吸引力大小本身非常接近 21 个精氨酸电荷的预期值。然而，排斥力明显大于精氨酸肽测量的预期，这表明鱼精蛋白中存在的中性氨基酸导致残余排斥力增加。我们通过测量合成六精氨酸的 DNA 力曲线证实了这一假设，其中合成的六精氨酸掺入了越来越多的中性氨基酸 我们还测量了鲑鱼精子核中的 DNA 组装力。体内精子和体外重组的 DNA-鲑鱼鱼精蛋白力实际上是相同的。因此，这些力测量具有直接的生物学应用。我们可以利用 DNA 组装力的知识来研究精子头部 DNA 包装、DNA 损伤和男性不育之间的联系。文献中有几篇报道将人类男性不育症与鱼精蛋白异常联系起来。由于精子中缺乏 DNA 修复系统，活性氧造成的累积 DNA 损伤是导致男性不育的主要直接因素。我们假设，由于鱼精蛋白不足或改变而导致的 DNA 组装不正确，将导致精子中 DNA 包装不紧密（螺旋之间的间距大于正常间距），从而使 DNA 更容易暴露于氧化自由基。我们可以通过直接测量正常和不育男性精子螺旋之间的距离以及累积的 DNA 损伤来检验这一假设。