NONCOVALENT INTERMOLECULAR INTERACTIONS IN BIOCHEMISTRY

生物化学中的非共价分子间相互作用

• 批准号: 6289725
• 负责人: Allen P Minton
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 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/6289725
• 关键词: analytical method; biomedical equipment development; chemical kinetics; fiber optics; intermolecular interaction; macromolecule; mathematical model; membrane transport proteins; method development; proteins; receptor binding; sedimentation equilibrium; spectrometry; tryptophan synthase

Statistical thermodynamic and kinetic models have been developed to describe rates and equilibria of reversible adsorption of proteins to surfaces under conditions such that molecules of adsorbed protein may interact with each other via short range steric repulsion and nonspecific attraction leading to the formation of adsorbate clusters. It is found that if attractive forces are sufficiently strong, they may counteract the highly negatively cooperative effect of steric repulsion, leading to equilibrium adsorption isotherms and adsorption progress curves that are as steep as or steeper than those calculated from simple Langmuir (non-cooperative) adsorption theory, in agreement with a variety of experimental observations. The results obtained are qualitatively independent of the assumed shape of the clusters, providing that clusters are represented as convex bodies. The dispersity, size, shape, and self-interaction of generation 5 polyamidoamine dendrimeric polymers with amino- and hydroxyl terminal groups (surfaces) were characterized using several physicochemical techniques. It was found that amino-surface dendrimers form oligomeric aggregates in aqueous solution, even in the presence of high salt concentrations (0.6 M sodium phosphate). In contrast, the hydroxyl- surface polymer G5-OH behaves as a single homogeneous (or paucidisperse) species at low concentration. Measurements of density increment and the sedimentation and diffusion coefficients of G5-OH suggest a more swollen, porous structure than a globular protein of comparable mass. Measurements of the concentration dependence of sedimentation equilibrium of G5-OH in pH 7.2 phosphate buffer indicate the presence of significant electrostatic repulsion overlaid on weakly attractive interactions, leading to the formation of nonspecific aggregates at sufficiently high dendrimer concentration. The spectrophotometric absorbance scanner reported as under construction in last years annual report has been completed and is currently undergoing testing and fine-tuning to achieve the anticipated accuracy and precision.The technique of nonideal tracer sedimentation equilibrium recently developed in our laboratory has been used to measure the effect of high concentrations of an inert polymer, dextran, on the state of association of calf brain tubulin under nonassembly conditions (i.e., under conditions in which large arrays of tubulin, such as microtubules, are not formed). It was found that with increasing dextran concentration, the weight-average molar mass of tubulin increases progressively to several times that of the monomer, indicating the presence of soluble tubulin oligomers which are believed to constitute precursors to the formation of large insoluble arrays. The self-association of the bacterial cell division protein FtsZ has been studied via sedimentation equilibrium. The dependence of the weight-average molar mass on total protein concentration has been measured as a function of the concentration of magnesium ion. Analysis of the data indicates that under the conditions of the experiments, FtsZ forms indefinitely large aggregates with a stepwise equilibrium association constant that decreases slightly with increasing aggregate size, approaching an asymptotic limit. The dependence of the asymptotic limit upon Mg concentration indicates that one Mg ion is taken up for each molecule of FtsZ added to the growing aggregate. - protein adsorption, statistical thermodynamics, dendritic polymers, fiber-optic spectrophotometer, sedimentation equilibrium, protein association

统计热力学和动力学模型被用来描述蛋白质在表面的可逆吸附的速率和平衡，在这种条件下，被吸附的蛋白质分子可以通过短程空间排斥和非特异性吸引相互作用，从而形成吸附簇。结果表明，如果引力足够强，它们可以抵消空间位阻的高度负协同效应，导致平衡吸附等温线和吸附过程曲线与简单的朗缪尔(非合作)吸附理论计算的结果一样陡峭或更陡峭，这与各种实验观测结果一致。假设团簇被表示为凸体，得到的结果定性地独立于所假定的团簇形状。用多种物理化学方法表征了具有氨基和羟基端基(表面)的第5代聚酰胺胺树枝状聚合物的分散性、大小、形状和自相互作用。研究发现，即使在高盐浓度(0.6M磷酸钠)存在的情况下，氨基表面树枝状大分子也能在水溶液中形成低聚聚集体。相反，羟基表面聚合物G5-OH在低浓度时表现为单一的均匀(或稀有分散)物种。密度增量以及G5-OH的沉淀和扩散系数的测量表明，与同等质量的球状蛋白质相比，G5-OH具有更膨胀、更多孔的结构。对G5-OH在pH 7.2磷酸盐缓冲液中的沉降平衡浓度依赖关系的测量表明，在弱吸引相互作用上存在显著的静电排斥，导致在足够高的树枝状大分子浓度下形成非特异性聚集体。去年年报中报告在建的分光光度扫描仪已经完成，目前正在进行测试和微调，以达到预期的准确度和精密度。本实验室最近开发的非理想示踪剂沉积平衡技术被用于测量在非组装条件下(即在没有形成大量微管等微管的条件下)高浓度的惰性聚合物葡聚糖对小牛脑微管蛋白结合状态的影响。研究发现，随着葡聚糖浓度的增加，微管蛋白的重均摩尔质量逐渐增加到单体的数倍，这表明存在可溶的微管蛋白低聚物，它们被认为是形成大的不溶阵列的前体。用沉降法研究了细菌细胞分裂蛋白FtsZ的自缔合作用。重均摩尔质量对总蛋白质浓度的依赖关系已被测量为镁离子浓度的函数。对实验数据的分析表明，在实验条件下，FtsZ形成了无限大的团聚体，具有逐步平衡的缔合常数，随着团聚体尺寸的增加而略有减小，接近一个渐近极限。渐近极限与镁浓度的关系表明，添加到不断增长的聚集体中的FtsZ分子每吸收一个镁离子。-蛋白质吸附、统计热力学、树枝状聚合物、光纤分光光度计、沉淀平衡、蛋白质缔合