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.

真核核，精子核，病毒和细菌中的致密包装DNA对于适当的细胞功能是必需的。通过多价离子组装DNA是一个关键的测试基础，不仅了解DNA的体内压实，而且还了解带电分子之间相互作用的物理学。如果存在足够浓度的多价离子，DNA将自发地组装成有序的阵列。螺旋不会塌陷到触摸，但根据冷凝离子的性质而被0.5-1.5 nm的溶剂分离。在平衡间距处有吸引力和排斥力平衡。通过将渗透应力结合在一起，将实验与单分子，磁镊子，拉动实验一起推动，我们能够在几种常用的凝位剂的平衡间距处将有吸引力和排斥的自由能分开。结果证实了我们先前关于水合力的假设。有0.25 nm的衰减长度指数的排斥力，在静电剂中相当于图像电荷排斥力的水合。从溶剂表面延伸的水合气氛稳定了表面的水结构。通过用另一个表面代替水来破坏大气，无论另一个表面上的水结构如何，都将降低水合能。排斥应主要依赖于DNA表面上的基团的水结构，也许应取决于结合模式（磷酸骨架或凹槽），但不取决于这些基团与螺旋螺旋的相关性。该吸引力还具有指数力的特征，但具有排斥力的衰减长度（0.5 nm）的两倍。吸引力是由表面水合结构的直接相互作用引起的。由于另一个表面的近距离存在，水结构的扰动可以根据相互结构水的不同而削弱或增强水合能量。我们假设，吸引力具有与先前对排斥水的指数衰减长度相同的指数0.5 nm衰减长度，但是由于辅助螺旋的互补水结构的相关性，该力现在具有吸引力。通过进一步研究长度不同的生物烷基胺和精氨酸肽，我们确定了吸引力的大小随着电荷的数量而增加，而与单个分子相关的熵损失一致，而相互作用的增加，但相互作用的增益与费用数量增加。排斥力仅略微依赖于预期的电荷数量，但确实取决于结合柜台的化学性质。 我们现在正在研究组装DNA的鲑鱼精蛋白的包装力。精蛋白是小精氨酸的小肽，用于包装精子头中的DNA。精素-DNA力类似于聚精氨酸-DNA相互作用。然而，DNA螺旋之间的平衡间距对应于四方或五体精氨酸，而不是实际存在的21个精氨酸电荷。我们已经将精蛋白DNA力分离为它们的吸引力和排斥成分。基于我们先前对精氨酸肽的测量值，吸引力的幅度本身非常接近21个精氨酸电荷的预期。然而，排斥力明显大于精氨酸肽测量值的预期，这表明精神素中存在的中性氨基酸会导致残留抑制增加。我们已经通过用合成六角碱测量DNA力曲线来证实这一假设，该曲线融合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，重构的DNA-salmon精蛋白原力实际上是相同的。因此，这些力测量具有直接的生物应用。我们可以利用对DNA组装力的知识来研究精子头，DNA损伤和男性不育症中的DNA包装之间的联系。文献中有几份报道将人类不育症与精蛋白异常相关。由于精子中不存在DNA修复系统，因此由于活性氧引起的累积DNA损伤是男性不育症的主要贡献者。我们假设通过不足或改变精蛋白改变DNA的不正确组装将导致精子中包装不太紧密的DNA（螺旋之间的间距比正常间距要大于正常的间距），因此，DNA可能会暴露于氧化自由基。我们可以通过直接测量正常雄性和不育男性和累积的DNA损伤的螺旋之间的距离进行直接测量来检验这一假设。