NONCOVALENT INTERMOLECULAR INTERACTIONS IN BIOCHEMISTRY

已经建立了统计热力学和动力学模型来描述蛋白质在表面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距离的空间排斥和非特异性的吸引相互作用，从而导致吸附物簇的形成。研究发现，如果引力足够强，它们可以抵消空间排斥力的高度负合作效应，导致平衡吸附等温线和吸附过程曲线与简单Langmuir（非合作）吸附理论计算的曲线一样陡峭或更陡峭，这与各种实验观察结果一致。所获得的结果在质量上独立于假定的簇的形状，提供簇被表示为凸体。利用多种物理化学技术表征了第5代氨基和羟基末端基团（表面）的聚氨基胺树胶聚合物的分散性、大小、形状和自相互作用。研究发现，即使在高盐浓度（0.6 M磷酸钠）的水溶液中，氨基表面的树状大分子也能形成低聚物聚集体。相反，羟基表面聚合物G5-OH在低浓度下表现为单一的均相（或低分散）物质。对G5-OH的密度增量、沉降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具有更肿胀的多孔结构。在pH值为7.2的磷酸盐缓冲液中，G5-OH沉降平衡的浓度依赖测量表明，在弱吸引相互作用上存在显著的静电斥力，导致在足够高的树突分子浓度下形成非特异性聚集体。在去年的年度报告中报告正在建造的分光光度吸收扫描仪已经完成，目前正在进行测试和微调，以达到预期的准确度和精密度。我们实验室最近开发的非理想示踪沉降平衡技术已被用于测量高浓度的惰性聚合物葡聚糖在非组装条件下（即在未形成大阵列微管等微管的条件下）对犊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，微管蛋白的重量平均摩尔质量逐渐增加到单体的数倍，表明存在可溶性微管蛋白低聚物，这些低聚物被认为是形成大的不溶性阵列的前体。通过沉降平衡法研究了细菌细胞分裂蛋白FtsZ的自结合。重量-平均摩尔质量对总蛋白浓度的依赖性作为镁离子浓度的函数进行了测量。数据分析表明，在实验条件下，FtsZ形成无限大的团聚体，其平衡关联常数随团聚体大小的增加而略有减小，接近渐近极限。渐近极限与Mg浓度的关系表明，每加入一个FtsZ分子，就会占用一个Mg离子。-蛋白质吸附，统计热力学，树突聚合物，光纤分光光度计，沉淀平衡，蛋白质缔合

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