Assembly Forces And Recognition Reactions

装配力和识别反应

• 批准号: 8149307
• 负责人: Donald Rau
• 金额: $ 36.28万
• 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/8149307
• 关键词: Arginine; Binding; Biological; Characteristics; Chemicals; Complex; Consensus; Cyclic AMP-Dependent Protein Kinases; DNA; DNA Damage; DNA Packaging; DNA Repair; DNA Sequence; Disease; Disulfides; Electrostatics; Entropy; Equilibrium; Free Radicals; Goals; Histones; Human; Hydration status; Image; In Vitro; Infertility; Ions; Knowledge; Length; Link; Lipid Bilayers; Literature; Male Infertility; Measurement; Metals; Methenamine; Nature; Neutral Amino Acids; Oligonucleotides; Osmotic Pressure; Peptides; Phosphoric Monoester Hydrolases; Phosphorylated Peptide; Phosphorylation; Physics; Protamines; Protein Binding; Protein Dephosphorylation; Proteins; Protocols documentation; Reaction; Reactive Oxygen Species; Reducing Agents; Residual state; Role; Salmon; Salts; Sequence-Specific DNA Binding Protein; Series; Serine; Sodium Chloride; Solutions; Solvents; Specificity; Sperm Head; Spermatogenesis; Spermidine; Spermine; Stress; Surface; Testing; Virus; Water; Work; in vivo; macromolecule; male; nanometer; planetary Atmosphere; prevent; sperm cell

The ability to measure directly forces between biopolymers in macroscopic condensed arrays has greatly changed our understanding of how molecules interact at close spacings, the last 1-1.5 nanometers separation. The universality of the force characteristics observed for a wide variety of macromolecules, including DNA, proteins, lipid bilayers, and carbohydrates, and for the exclusion of small solutes and salts from polar and nonpolar macromolecular surfaces has led us to conclude that the energy associated with changes in structuring water between surfaces dominates intermolecular forces. 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. We have been able to separate the attractive and repulsive free energies at the equilibrium spacing for the several commonly used condensing agents and for a series of homologous oligo-arginine peptides. 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. 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 a set of oligo-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 0.25 nm decay length repulsive force is only slightly dependent on the number of charges, but does depend on the chemical nature of the bound counterion. We have extended this work and are now investigating condensed arrays of protamine assembled DNA. Protamines are small (30-50 aa), arginine-rich (60-65%) peptides used to package DNA in sperm heads. Significantly, protamine-DNA forces resemble the forces seen for all other condensing ions we have measured, from simple metal ions as Mn2+ and Co3+ to the biogenic amines spermidine and spermine to the oligo-arginine peptide series. There seems to be no additional contribution from the protein character of protamines. The repulsive force, however, is significantly larger than expected from the oligo-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. Surprisingly the character of the neutral amino acid from hydrophilic serines to hydrophobic isoleucines makes little difference to the overall force magnitude. We have also measured DNA assembly forces in salmon sperm nuclei. The in vivo sperm and in vitro, reconstituted salmon protamine - DNA forces are virtually identical. These force measurements thus have direct biological application. Mammalian protamines are more complicated than piscine; two protamine species are typically present and disulphide bridges form between protamines presumably to further condense DNA. This can be easily verified by measuring DNA-DNA spacing with and without added disulphide reducing agents. We will optimize our experimental protocols with bull sperm before investigating human sperm. 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 contributor to male infertility since DNA repair systems are absent in sperm. We hypothesize that incorrect assembly of DNA by insufficient or modified protamines will result in less tightly packaged DNA in sperm (a larger than normal distance between helices) and, consequently, a greater accessibility of oxidizing free radicals to DNA. 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. The replacement of histones by protamines in spermatogenesis occurs in several steps. Before binding to DNA, the several of the protamine serines are first phosphorylated. It is only afterwards that phosphorylated protamine- DNA complexes are dephosphorylated and the DNA fully packaged. We have examined the effect of phosphorylation of synthetic protamine-like peptides on DNA-DNA forces. As expected from previous measurements, R3SR3 is only slightly different from R6. The phosphorylated peptide, R3pSR3, however, profoundly weakens attraction, increasing the distance between DNA surfaces from 0.85 nm to 1.35 nm even in the absence of any competing salt as would be found in the cell. This level of phosphorylation mimics that found in naturally occurring protamines. This greatly weakened attraction suggests that the function of protamine phosphorylation is to prevent tight packaging of DNA and to allow phosphatase access. An orderly dephosphorylation of these modified protamines would consequently allow an orderly condensation of DNA. Incomplete dephosphorylation and, therefore, less tightly assembled DNA has been linked to infertility. We are currently phosphorylating Clupine protamine using protein kinase A. This particular protamine has three consensus PKA sequences for serine phosphorylation. We have also investigated the additivity of DNA-DNA forces with mixed ions. The force magnitudes of both the 2.5 and 5 Angstrom decay length forces vary with the kind of ion bound to DNA. This ion specificity depends on charge or water organizing ability and binding location on DNA. Bound ions may interact synergistically to maximize attraction. This knowledge is important for predicting interaction energies in real biological systems. To this end, the forces in cobalt hexamine+3, Mg2+, and putrescine2+ were separately and carefully measured and then parameterized. Forces were then measured in 1 mM Co3+ with concentrations of Mg2+ or putrescine2+ varied from 4 to 80 mM. The fraction of DNA charge neutralized with Co3+ was determined spectrophotometrically. To a good first order approximation, the observed forces were simply a linear combination of the contributions from each of the bound ions.

宏观冷凝阵列中生物聚合物之间直接作用的力的能力极大地改变了我们对分子如何在近距离之间相互作用的理解，即最后的1-1.5纳米分离。观察到的各种大分子的力特征的普遍性，包括DNA，蛋白质，脂质双层和碳水化合物，以及排除来自极性和非极性大分子表面的小溶质和盐，使我们得出结论，可以得出结论，该能量与我们的能量相关。在表面之间结构水的变化主导分子间力。 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。我们已经能够在几种常用的冷凝剂和一系列同源的寡精氨酸肽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。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。通过进一步研究一组不同长度的寡素 - 精氨酸肽，我们确定吸引力的幅度随着电荷的数量而增加，与熵持续损失相关的电荷数量，无论与电荷相关，而相互作用的增加，但相互作用的增长能量随着电荷的数量而增加。 0.25 nm的衰减长度排斥力仅略微依赖于电荷的数量，但确实取决于结合柜台的化学性质。 我们已经扩展了这项工作，现在正在研究精蛋白组装的DNA的冷凝阵列。精蛋白小（30-50 AA），精氨酸富含精氨酸（60-65％）的肽用于包装精子头中的DNA。值得注意的是，精氨酸-DNA力类似于我们测量的所有其他冷凝离子所见的力，从简单的金属离子为MN2+和Co3+到生物胺精子和杀生物胺的生物氨基胺序列。蛋白质特征似乎没有其他贡献。然而，排斥力明显大于寡氨酸肽测量值的预期，这表明精蛋白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hexa-精氨酸来测量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。令人惊讶的是，中性氨基酸从亲水丝氨酸到疏水性异酸的特征对整体力量幅度几乎没有影响。 我们还测量了鲑鱼精子核中的DNA组装力。体内精子和体外，重建的鲑鱼精蛋白 - DNA力实际上是相同的。因此，这些力测量具有直接的生物应用。哺乳动物的蛋白质比piscine更复杂。通常存在两种精蛋白种类，并且在精蛋白之间形成了二硫键，大概是为了进一步凝结DNA。可以通过测量和不增加二硫化物还原剂的DNA-DNA间距来轻松验证。在研究人类精子之前，我们将用牛精子优化我们的实验方案。 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因素。我们假设通过不足或修饰的精蛋白将DNA的组装不正确会导致精子（螺旋之间的距离比正常距离大于正常距离）的紧密包装的DNA，因此，氧化自由基对DNA的氧化更大的可及性。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。 精蛋白在精子发生中替代了组蛋白，以多个步骤发生。在与DNA结合之前，首先将几种精蛋白丝氨酸磷酸化。仅此后，将磷酸化的精蛋白DNA复合物被去磷酸化并完全包装DNA。我们已经检查了合成精蛋白样肽磷酸化对DNA-DNA力的磷酸化的影响。如前所述，R3SR3与R6仅略有不同。然而，即使在没有任何竞争盐的情况下，磷酸化的肽R3PSR3也深刻地削弱了吸引力，即使在没有任何竞争盐的情况下，DNA表面之间的距离也从0.85 nm增加到1.35 nm。这种磷酸化的水平模仿在天然出现的蛋白酶中发现。这种大大削弱的吸引力表明，精素磷酸化的功能是防止DNA的紧密包装并允许磷酸酶进入。因此，这些修饰的精选的有序去磷酸化将允许有序的DNA凝结。因此，不完全的去磷酸化，因此组装不足的DNA与不育有关。我们目前使用蛋白激酶A磷酸化的胶状精蛋白A磷酸化。该特定的精蛋白具有三个共有的PKA序列用于丝氨酸磷酸化。 我们还研究了使用混合离子的DNA-DNA力的添加性。 2.5和5埃埃斯特罗姆衰减长度的力幅度随与DNA结合的离子的种类而变化。该离子特异性取决于电荷或水的组织能力和DNA上的结合位置。绑定离子可以协同交互以最大化吸引力。这些知识对于预测实际生物系统中的相互作用能很重要。为此，分别并仔细测量了钴胺+3，mg2+和putrescine2+中的力，然后进行参数化。然后将力在1 mM CO3+中测量，浓度为Mg2+或Putrescine2+从4到80 mm不等。通过分光光度法确定用CO3+中和的DNA电荷的分数。在良好的一阶近似值中，观察到的力只是每个结合离子的贡献的线性组合。