Noncovalent Intermolecular Interactions In Biochemistry

本实验室新近发展的非理想示踪沉降平衡技术已被用来测量高浓度的影响（至多150 g/l）的血红蛋白（Hb）或牛血清白蛋白（BSA）两种蛋白质中的任一种对细菌细胞分裂蛋白FtsZ在不存在Mg离子的情况下的热力学活性和/或缔合状态的影响，其中FtsZ不自缔合，在Mg的存在下，FtsZ自缔合形成线性棒状低聚物的非均相混合物（见去年的报告）。结果发现，在镁的情况下，在存在的Hb或BSA的FtsZ的行为可以占定量的假设，单体FtsZ和BSA或Hb之间的相互作用是纯粹的空间排斥，并可以定量描述的模型，其中每个物种是由一个等效的硬球的大小约等于实际的蛋白质分子。还发现在Mg的存在下，FtsZ自我的程度对于给定的总浓度的FtsZ，缔合随着Hb或BSA浓度的增加而显著增加，其方式由排除体积理论定量描述，用于其中FtsZ的寡聚体由具有近似等于单体FtsZ的半径的固定半径和由FtsZ的守恒确定的长度的球形圆柱体表示的模型。马萨诸塞州 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的函数，包括蛋白质的浓度、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，研究了微管蛋白大阵列组装的速率和平衡。很明显，“惰性”添加剂的加入大大加速并增加了阵列形成的平衡程度，定性雅阁排除体积理论的预测。进一步的实验和制定的定量模型的组装过程正在进行中。 DNA结合蛋白，CAP，和一个42碱基对的寡核苷酸含有一个特定的CAP序列之间的特定复合物的形成，已经研究了示踪沉降平衡在不存在和存在的高浓度的“惰性”蛋白质，试图辨别的影响，惰性蛋白质的CAP的亲和力的寡核苷酸。目前正在为这一进程制定量化模型。 葡聚糖，一种惰性的水溶性聚合物，对载脂蛋白CII的淀粉样纤维的形成速率的影响已通过时间依赖性比浊法和时间依赖性的聚合物造粒进行了研究。发现以高达150 g/l的浓度添加聚合物以可以通过简单的排除体积模型定量描述的方式将淀粉样蛋白形成的相对速率增加高达8倍。 通过吸光度的温度依赖性和近、远紫外圆二色性研究了右旋糖酐对溶菌酶热变性稳定性的影响。当葡聚糖浓度增加到300 g/l时，发现半变性温度有小但显著的增加。旨在阐明这种效应的基础的进一步研究正在进行中。

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.