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.

精氨酸富含精氨酸的肽（30-50个氨基酸）自发地将DNA组装成延伸的，封闭的（<1 nm的地表到表面分离），液晶阵列，适合通过X射线散射来表征。重构的精蛋白-DNA组件和精子核具有非常相似的X射线散射曲线。使用合成肽，我们能够确定（1）香气几乎仅在赖氨酸上使用精氨酸来凝结DNA，因为相比之下，富含赖氨酸的肽填充DNA非常差，并且（2）鲑鱼精子核中的DNA填料密度完全由21精氨酸和33％中性氨基氨基氨基蛋白的中性氨基酸完全确定。 辅助生殖技术（ART）通常使用精子的密度分馏，以确保仅使用高质量的精子来人为地授精OVA。鲑鱼精子核可以在蔗糖密度梯度上分馏。较密集的馏分具有狭窄的DNA间旋转距离分布，峰值在0.9 nm的表面向表面分离处。密度较小的分数具有相同的峰间距，但是散射曲线偏向较大的分离，表明DNA亚群更松散。在光分数中添加多余的精蛋白可恢复密度分数的散射曲线。密度较少的精子核的偏斜散射曲线是由于精蛋白不足所致。 精神素不足会影响重构的精蛋白-DNA组件中的DNA填料与精子核重构的组件不同，只有90％的精蛋白可以中和DNA所需的精蛋白显示出散射峰，这简单地将散射峰转移到较大的间距离上，而不是将散射散射。我们假设精蛋白可以在重构的凝结物中平衡，但不能在精子核中平衡。这表明精子核中的DNA被组织成可预防精蛋白平衡的离散结构域。 （101）反射是室内和​​螺旋重复散射的组合。它的幅度在很大程度上取决于相邻螺旋的磷酸盐的相关性。 （101）反射的强度相对于重构的组件和精子核之间的主要内部反射存在明显差异。核（101）反射的强度相对损失与精子核中DNA的可疑环形堆积一致。环形DNA强烈弯曲，破坏了磷酸磷酸盐的相关性。这些怀疑的环形线也可能是阻碍精神蛋白在整个细胞核中平衡的结构域。我们正在使用核酸酶消化探测可能的域结构。 在替换DNA上的组蛋白时，精蛋白最初是丝氨酸磷酸化的。从精子中分离出的鲑鱼精蛋白中约有2-5％仍被磷酸化。不完全的去磷酸化与DNA损伤增加有关。我们以前已经发现，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。基于简单的净电荷减少，远远超出了预期的范围。可以用蛋白激酶A部分磷酸化精神。我们发现精蛋白磷酸化确实会大大降低螺旋之间的吸引力。即使将精蛋白电荷减少5％的适中磷酸化也可以将氧化物种可访问的体积增加50％。 我们对DNA螺旋之间的力的直接测量表明，水结构在近距离占据相互作用。这些水合力显示了两个组成部分：较长的指数力，衰减长度为0.5 nm，这是由于一个螺旋在一个螺旋上与邻近螺旋上的水合结构直接相互作用。根据两个螺旋上的水结构的互补程度，这种力可以具有排斥性或吸引力。第二个总是以衰减长度一半的排斥指数力是静电图像电荷排斥的水合力。力振幅不仅必须反映DNA本身的水合，还反映了DNA缩合柜台周围的水结构。我们先前已经解析了氨基酸对精蛋白DNA组件的填料密度的贡献。我们发现了控制力振幅的相当简单的规则。毫不奇怪，远距离吸引力随肽中精氨酸的数量而增加，而短距离排斥只会略有影响。相比之下，中性氨基酸的包含会增加短距离排斥，同时最小化吸引吸引力。我们假设中性氨基酸从DNA中移位出来，而没有显着影响精氨酸-DNA磷酸磷酸盐的吸引力。这种流离失所的水会增加水合图像样排斥。 现在，我们研究了一组同源单和二价烷基胺柜台，以进一步连接水合和力振幅。将第一个甲基添加到铵（H3N-CH3+）中增加了远程排斥，而对水合图像样排斥的影响很大。这种取代减少了氢供体的数量，并减少了离子的水合。现在，将第二个烷基碳添加到第一烷基碳（H3N+-CH2-CH3）现在几乎没有对远距离排斥的影响，但大大增加了短距离，半末长的排斥力。添加另一个碳群（H3n+ - （CH22-CH3）遵循相同的趋势。氢键供体的数量保持恒定，但烷基的水从DNA中移动，烷基增加了图像的排斥。将甲基类似于甲基，直接添加到氮基因（H2n+（CH3）2和N+（CH3）2和N+（CH3）2和N+（CH3）2和N+（CH3）4）的水中，这是水分的数量和数量的数量，这些数字是水的数量，该数量是水的数量，该数量是水的数量，该数字是数量的。从DNA中置换水。 虽然单价烷基胺遵循精氨酸中性氨基酸系列的趋势，但二价烷基胺显示出截然不同的行为。我们研究了 + NH2-（CH2）M-NH2 +的系列，M = 4和5二氨基分别是生物胺，perrescine和Cadaverine。在这种情况下，烷基接头长度对短距离半末长的长度没有影响，但是较长范围的力的排斥幅度随接头长度线性增加。单胺和二胺之间烷基作用的差异表明，力幅度不仅取决于反柜的化学性质，还取决于结合模式，即领土，磷酸盐或凹槽。 在与Alex Evilevitch的合作中，我们发现了一种新颖的过渡到更流动的DNA状态，在噬菌体兰巴达和人类单纯疱疹病毒1型中都紧密包装了。这两种病毒的过渡发生在约35oC，接近自然温度以供感染。过渡后DNA的流体状状态较高，它会使DNA更容易地将DNA射入目标细胞中。