Noncovalent Intermolecular Interactions In Biochemistry

生物化学中的非共价分子间相互作用

• 批准号: 6507261
• 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/6507261
• 关键词: DNA binding protein; amyloid proteins; apolipoproteins; bacterial proteins; biochemistry; bovine serum albumin; cell cycle proteins; chemical association; chemical kinetics; circular dichroism; dextrans; hemoglobin; intermolecular interaction; macromolecule; magnesium; microtubules; oligonucleotides; polymers; protein denaturation; sedimentation equilibrium; spectrometry; temperature; thermodynamics; tubulin

The technique of nonideal tracer sedimentation equilibrium recently developed in our laboratory has been used to measure the effect of high concentrations (up to 150 g/l) of either of two proteins, hemoglobin (Hb) or bovine serum albumin (BSA), on the thermodynamic activity and/or state of association of the bacterial cell division protein FtsZ in the absence of Mg ion, where FtsZ does not self-associate, and in the presence of Mg, where FtsZ self-associates to form a heterogeneous mixture of linear rodlike oligomers (see last year's report). It was found that in the absence of Mg, the behavior of FtsZ in the presence of either Hb or BSA may be accounted for quantitatively by the assumption that interactions between monomeric FtsZ and either BSA or Hb are purely steric-repulsive, and may be quantitatively described by models in which each species is represented by an equivalent hard sphere with a size approximately equal to that of the actual protein molecule. It was also found that in the presence of Mg, the extent of FtsZ self-association for a given total concentration of FtsZ increases substantially with increasing concentration of either Hb or BSA in a manner that is quantitatively described by excluded volume theory for a model in which oligomers of FtsZ are represented by spherocylinders having a fixed radius approximately equal to that of monomeric FtsZ and a length determined by conservation of mass. The rates and equilibria governing the assembly of large arrays of tubulin were studied via measurement of the wavelength-dependent turbidity of tubulin solutions as a function of time and various experimental variables, including the concentration of protein, the concentration and type of guanine nucleotides, pH, buffer salts, temperature, and the concentration of various "inert" additives. It is evident that addition of "inert" additives greatly accelerates and increases the equilibrium extent of array formation in qualitative accord with predictions of excluded volume theory. Further experimentation and formulation of quantitative models of the assembly process are in progress. The formation of a specific complex between an DNA-binding protein, CAP, and a 42 base-pair oligonucleotide containing a sequence specific for CAP, has been studied by tracer sedimentation equilibrium in the absence and presence of high concentrations of "inert" proteins in an attempt to discern the effect of the inert proteins on the affinity of CAP for the oligonucleotide. Quantitative models for this process are being formulated. The effect of dextran, an inert water-soluble polymer, on the rate of formation of amyloid fiber by apolipoprotein CII has been studied by time-dependent turbidimetry and time-dependent pelleting of polymer. It was found that addition of polymer at concentrations of up to 150 g/l increases the relative rate of amyloid formation by up to eight-fold in a manner that may be described quantitatively by a simple excluded volume model. The effect of dextran on the stability of lysozyme with respect to thermal denaturation has been studied via temperature dependence of absorbance and near and far uv circular dicrhroism. A small but significant increase in the half-denaturation temperature is found upon increasing dextran concentration to 300 g/l. Further studies aimed at elucidating the basis of this effect are underway.

我们实验室最近开发的非理想示踪沉降平衡技术已用于测量两种蛋白质（血红蛋白 (Hb) 或牛血清白蛋白 (BSA)）的高浓度（高达 150 g/l）对在不存在 Mg 离子的情况下细菌细胞分裂蛋白 FtsZ 的热力学活性和/或缔合状态的影响，其中 FtsZ 不存在 自缔合，并且在 Mg 存在的情况下，FtsZ 自缔合形成线性棒状低聚物的异质混合物（参见去年的报告）。研究发现，在不存在 Mg 的情况下，在存在 Hb 或 BSA 的情况下，FtsZ 的行为可以通过假设单体 FtsZ 与 BSA 或 Hb 之间的相互作用纯粹是空间排斥来定量解释，并且可以通过模型定量描述，其中每个物种由大小约等于实际蛋白质分子的等效硬球代表。还发现，在存在 Mg 的情况下，对于给定的 FtsZ 总浓度，FtsZ 自缔合的程度随着 Hb 或 BSA 浓度的增加而显着增加，其方式通过模型的排阻体积理论定量描述，其中 FtsZ 低聚物由具有约等于单体 FtsZ 的固定半径和由质量守恒确定的长度的球柱表示。 通过测量微管蛋白溶液的波长依赖性浊度作为时间和各种实验变量（包括蛋白质浓度、鸟嘌呤核苷酸的浓度和类型、pH、缓冲盐、温度和各种“惰性”添加剂的浓度）的函数，研究了控制大微管蛋白阵列组装的速率和平衡。显然，“惰性”添加剂的添加极大地加速并增加了阵列形成的平衡程度，这与排除体积理论的预测定性一致。组装过程的定量模型的进一步实验和制定正在进行中。 在不存在和存在高浓度“惰性”蛋白的情况下，通过示踪剂沉降平衡研究了 DNA 结合蛋白 CAP 和含有 CAP 特异性序列的 42 个碱基对寡核苷酸之间的特异性复合物的形成，试图辨别惰性蛋白对 CAP 对寡核苷酸的亲和力的影响。正在制定该过程的定量模型。 葡聚糖（一种惰性水溶性聚合物）对载脂蛋白 CII 形成淀粉样纤维的速率的影响已通过时间依赖性比浊法和聚合物的时间依赖性造粒进行了研究。结果发现，添加浓度高达 150 g/l 的聚合物可使淀粉样蛋白形成的相对速率增加高达八倍，其方式可以通过简单的排除体积模型定量描述。 通过吸光度的温度依赖性以及近、远紫外圆二色性研究了右旋糖酐对溶菌酶在热变性方面的稳定性的影响。当葡聚糖浓度增加到 300 g/l 时，发现半变性温度有小幅但显着的增加。旨在阐明这种效应的基础的进一步研究正在进行中。