Assembly Forces And Recognition Reactions

装配力和识别反应

• 批准号: 8941486
• 负责人: Donald Rau
• 金额: $ 45.33万
• 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/8941486
• 关键词: Affect; Amines; Amino Acids; Ammonium; Arginine; Assisted Reproductive Technology; Bacteriophage lambda; Behavior; Binding; Biogenic Amines; Biological; Cadaverine; Capsid Proteins; Carbon; Cell Nucleus; Cell physiology; Cells; Cellular biology; Charge; Chemicals; Collaborations; Complex; Congenital Abnormality; Coupling; Cyclic AMP-Dependent Protein Kinases; DNA; DNA Damage; DNA Packaging; DNA Repair; Defect; Diamines; Digestion; Disease; Electrostatics; Ensure; Fractionation; Functional disorder; Genome; Goals; Herpesvirus 1; Histones; Human; Hydration status; Hydrogen; Hydrogen Bonding; Image; Infection; Infertility; Ions; Knowledge; Length; Light; Link; Liquid substance; Lysine; Measurement; Measures; Mono-S; Mutagens; Nature; Neutral Amino Acids; Nitrogen; Ovum; Peptides; Phosphorylation; Physics; Protamines; Protein Dephosphorylation; Proteins; Putrescine; Reaction; Relative (related person); Salmon; Series; Serine; Solutions; Specificity; Spontaneous abortion; Stress; Structure; Sucrose; Surface; Temperature; Virus; Water; alkyl group; aqueous; base; density; design; inorganic phosphate; macromolecule; methyl group; novel; nuclease; nucleic acid structure; prevent; protamine 1; reconstitution; sperm cell; sperm quality; structural biology; synthetic peptide; theories; trend

Arginine rich protamines peptides (30-50 amino acids) spontaneously assemble DNA into extended, close-packed (<1 nm surface-to-surface separation), liquid crystalline arrays that are suitable for characterization by x-ray scattering. Reconstituted protamine-DNA assemblies and sperm nuclei give very similar x-ray scattering profiles. Using synthetic peptides we were able to determine that (1) protamines use arginine almost exclusively over lysine to condense DNA because lysine rich peptides pack DNA very poorly in comparison and (2) the DNA packing density in salmon sperm nuclei is completely determined by the 21 arginines and 33% neutral amino acids of salmon protamine. Assisted reproductive technologies (ART) routinely use density fractionation of sperm to ensure only high quality sperm are used to artificially inseminate ova. Salmon sperm nuclei can be fractionated on a sucrose density gradient. The denser fraction has a narrow distribution of DNA interhelical distances with a peak at 0.9 nm surface-to-surface separation. The less dense fraction has the same peak spacing, but the scattering profile is skewed toward larger separations indicating a DNA subpopulation that is more loosely packed. Adding excess protamine to the light fraction recovers the scattering profile of the denser fraction. The skewed scattering profile of the less dense sperm nuclei is due to insufficient protamine. Insufficient protamine affects DNA packing in reconstituted protamine-DNA assemblies differently from sperm nuclei Reconstituted assemblies with only 90% of the protamine necessary to neutralize DNA show a scattering profile that simply shifts the scattering peak to larger interhelical distances rather than skewing the scattering profile. We hypothesize that protamines can equilibrate throughout a reconstituted condensate, but not in sperm nuclei. This would suggest that the DNA in sperm nuclei is organized into discrete domains that prevent protamine equilibration. The (101) reflection is a combination of interaxial and helical repeat scattering. Its amplitude strongly depends on the correlation of phosphates on adjacent helices. There is a distinct difference in the intensities of the (101)reflections relative to the main interaxial reflection between reconstituted assemblies and sperm nuclei. The relative loss of intensity of the (101) reflection with nuclei is consistent with the suspected toroidal packing of DNA in sperm nuclei. Toroidal DNA is strongly curved, disrupting phosphate-phosphate correlation. These suspected toroids may also be the domains that hinder the equilibration of protamine throughout the nucleus. We are probing a possible domain structure using nuclease digestion. Protamines are initially serine phosphorylated when replacing histones on DNA. About 2-5 % of salmon protamines isolated from sperm are still phosphorylated. Incomplete dephosphorylation has been linked to increased DNA damage. We have previously found that serine phosphorylation of synthetic arginine peptides greatly reduces the attraction between helices; far more than would be expected based on a simple reduction in net charge. Protamines can be partially phosphorylated with protein kinase A. We find that protamine phosphorylation does dramatically decrease the attraction between helices. Even modest phosphorylation that reduces the protamine charge by only 5% increases the volume accessible to oxidizing species by 50%. Our direct measurement of forces between DNA helices has shown that water structuring dominates interactions at close distances. These hydration forces show two components: a longer ranged exponential force with decay length 0.5 nm that is due to the direct interaction of the hydration structure on one helix with that on a neighboring helix. This force can be either repulsive or attractive depending on the extent of complementarity of water structuring on the two helices. A second always repulsive exponential force with half the decay length is the hydration force equivalent of electrostatic image charge repulsion. The force amplitudes necessarily reflect not only the hydration of DNA itself but also the water structuring around DNA condensed counterions. We have previously parsed the amino acid contributions to the packing density of protamine-DNA assemblies. We uncovered quite simple rules that govern force amplitudes. Not surprisingly, the long range attraction increases with number of arginines in the peptide, while the short range repulsion is only slightly affected. In contrast the inclusion of neutral amino acids increases the short range repulsion while minimally affecting the attraction. We postulated that the neutral amino acids displaced water from the DNA without significantly affecting the arginine-DNA phosphate attraction. This displaced water would increase the hydration image-like repulsion. We have now investigated a set of homologous mono- and di-valent alkyl amine counterions in order to further connect hydration and force amplitudes. The addition of the first methyl group to ammonium (H3N-CH3+) increases the long range repulsion without much effect on the hydration image-like repulsion. This substitution reduces the number of hydrogen donors and reduces hydration of the ion. Adding a second alkyl carbon to the first (H3N+-CH2-CH3) now has almost no effect on the long range repulsion, but greatly increases the short range, half-decay length repulsion. The addition of another carbon group (H3N+-(CH22-CH3) follows the same trend. The number of hydrogen bond donors remains constant, but waters are displaced from DNA by the alkyl groups increasing the image-like repulsion. Adding methyl groups directly to the nitrogen (H2N+(CH3)2 and N+(CH3)4) both reduces the number of hydrogen bond donors and, therefore, water structuring around these ions and displaces water from DNA. Both force amplitudes significantly. While the mono-valent alkyl amines followed the trend expected from the arginine-neutral amino acid series, the di-valent alkyl amines show quite different behavior. We investigated the series +NH2-(CH2)M-NH2+ for M ranging from 2 to 5. The M = 4 and 5 diamines are the biogenic amines, putrescine and cadaverine, respectively. In this case the alkyl linker length has no effect on the short range half-decay length force, but the repulsive amplitude of the longer ranged force increases linearly with linker length. The difference in the effect of alkyl groups between mono- and di- amines illustrates that force amplitudes depend sensitively on the not only on the chemical nature of the counterion but also on the binding mode, i.e., territorial, phosphate, or groove. In collaboration with Alex Evilevitch we have uncovered a novel transition to a more fluid-like state of DNA tightly packed in both the bacteriophage lambda and the human Herpes simplex virus type 1. Since a similar transition is not seen for DNA condensed from bulk solution, we postulate that the DNA bending energy from being condensed within a spherical protein capsid drives the transition. The transition occurs at about 35oC for both viruses, close to the natural temperature for infection. The more fluid-like state of the DNA after the transition would make ejection of DNA into the target cell easier.

富含精氨酸的鱼精蛋白肽（30-50 个氨基酸）自发地将 DNA 组装成延伸的、紧密堆积的（<1 nm 表面到表面分离）液晶阵列，适合通过 X 射线散射进行表征。重组的鱼精蛋白-DNA 组件和精子核给出了非常相似的 X 射线散射图。使用合成肽，我们能够确定 (1) 鱼精蛋白几乎完全使用精氨酸而不是赖氨酸来浓缩 DNA，因为相比之下，富含赖氨酸的肽包装 DNA 非常差，(2) 鲑鱼精子核中的 DNA 包装密度完全由 21精氨酸和鲑鱼鱼精蛋白的 33% 中性氨基酸。 辅助生殖技术 (ART) 通常使用精子的密度分级，以确保仅使用高质量的精子来对卵子进行人工授精。鲑鱼精子核可以在蔗糖密度梯度上分离。较致密的部分具有较窄的 DNA 螺旋间距分布，峰值位于 0.9 nm 表面与表面分离处。密度较低的部分具有相同的峰间距，但散射曲线偏向较大的间距，表明 DNA 亚群的堆积更加松散。向轻组分中添加过量的鱼精蛋白可恢复较致密组分的散射曲线。密度较低的精子核的倾斜散射曲线是由于鱼精蛋白不足造成的。 鱼精蛋白不足对重组鱼精蛋白-DNA 组装体中 DNA 堆积的影响与精子细胞核不同。重组组装体中只有 90% 的中和 DNA 所需的鱼精蛋白，显示出的散射曲线只是将散射峰移动到更大的螺旋间距离，而不是使散射曲线倾斜。我们假设鱼精蛋白可以在重构的冷凝物中平衡，但不能在精子核中平衡。这表明精子核中的 DNA 被组织成离散的结构域，从而阻止鱼精蛋白平衡。 (101) 反射是轴间散射和螺旋重复散射的组合。其幅度很大程度上取决于相邻螺旋上磷酸盐的相关性。相对于重建组件和精子核之间的主要轴间反射，(101)反射的强度存在明显差异。细胞核 (101) 反射强度的相对损失与精子细胞核中 DNA 的可疑环形堆积一致。环形 DNA 强烈弯曲，破坏了磷酸盐-磷酸盐的相关性。这些可疑的环形结构也可能是阻碍整个细胞核中鱼精蛋白平衡的区域。我们正在使用核酸酶消化来探索可能的结构域结构。 当取代 DNA 上的组蛋白时，鱼精蛋白最初被丝氨酸磷酸化。从精子中分离出的鲑鱼鱼精蛋白中约有 2-5% 仍处于磷酸化状态。不完全去磷酸化与 DNA 损伤增加有关。我们之前发现，合成精氨酸肽的丝氨酸磷酸化大大降低了螺旋之间的吸引力；远远超过基于净费用简单减少的预期。鱼精蛋白可以被蛋白激酶 A 部分磷酸化。我们发现鱼精蛋白磷酸化确实显着降低了螺旋之间的吸引力。即使是适度的磷酸化（仅将鱼精蛋白电荷减少 5%）也会使氧化物质可接触的体积增加 50%。 我们对 DNA 螺旋之间的力的直接测量表明，水的结构在近距离的相互作用中占主导地位。这些水合力显示出两个组成部分：衰减长度为 0.5 nm 的较长范围的指数力，这是由于一个螺旋上的水合结构与相邻螺旋上的水合结构的直接相互作用所致。该力可以是排斥力，也可以是吸引力，具体取决于两个螺旋上水结构的互补程度。第二个总是具有一半衰减长度的排斥指数力是相当于静电图像电荷排斥的水合力。力的幅度不仅必然反映 DNA 本身的水合作用，而且还反映 DNA 凝聚反离子周围的水结构。我们之前已经解析了氨基酸对鱼精蛋白-DNA 组装体堆积密度的贡献。我们发现了控制力幅值的非常简单的规则。毫不奇怪，长程吸引力随着肽中精氨酸的数量而增加，而短程排斥仅受到轻微影响。相反，包含中性氨基酸会增加短程排斥力，同时对吸引力的影响最小。我们假设中性氨基酸取代了 DNA 中的水，而没有显着影响精氨酸-DNA 磷酸盐的吸引力。这种排出的水会增加水合图像般的排斥力。 我们现在研究了一组同源的一价和二价烷基胺抗衡离子，以进一步连接水合和力振幅。在铵（H3N-CH3+）中添加第一个甲基会增加长程排斥力，而对水合图像排斥力没有太大影响。这种取代减少了氢供体的数量并减少了离子的水合作用。在第一个烷基碳 (H3N+-CH2-CH3) 上添加第二个烷基碳现在对长程斥力几乎没有影响，但大大增加了短程、半衰变长度斥力。另一个碳基团 (H3N+-(CH22-CH3) 的添加遵循相同的趋势。氢键供体的数量保持不变，但水被烷基基团从 DNA 中取代，增加了图像般的排斥力。直接添加甲基基团氮（H2N+(CH3)2 和 N+(CH3)4）都会减少氢键供体的数量，从而减少这些离子周围的水结构，并显着取代 DNA 中的水。 虽然一价烷基胺遵循精氨酸中性氨基酸系列预期的趋势，但二价烷基胺表现出截然不同的行为。我们研究了 M 范围从 2 到 5 的 +NH2-(CH2)M-NH2+ 系列。M = 4 和 5 二胺分别是生物胺、腐胺和尸胺。在这种情况下，烷基连接体长度对短程半衰变长度力没有影响，但较长程力的排斥幅度随连接体长度线性增加。单胺和二胺之间烷基作用的差异表明，力的振幅不仅敏感地取决于抗衡离子的化学性质，而且还取决于结合模式，即区域、磷酸盐或沟槽。 在与 Alex Evilevitch 的合作中，我们发现了在 lambda 噬菌体和人类 1 型单纯疱疹病毒中紧密堆积的 DNA 向更接近流体状态的转变。由于从大量溶液中浓缩的 DNA 没有观察到类似的转变，我们假设球形蛋白衣壳内凝聚的 DNA 弯曲能量驱动了这一转变。两种病毒的转变发生在 35oC 左右，接近感染的自然温度。转变后 DNA 的状态更加类似流体，这使得 DNA 更容易喷射到靶细胞中。