Noncovalent Intermolecular Interactions In Biochemistry

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（高达150 g/l）两种蛋白质中的任何一种，血红蛋白（Hb）或牛血清白蛋白（BSA），在没有Mg离子的情况下对细菌细胞分裂蛋白FtsZ的热力学活性和/或结合状态的影响，其中FtsZ不自结合，并且在Mg存在的情况下。其中FtsZ自结合形成线性棒状低聚物的异质混合物（见去年的报告）。研究发现，在没有Mg的情况下，FtsZ在Hb或BSA存在时的行为可以通过假设单体FtsZ与BSA或Hb之间的相互作用是纯粹的立体排斥来定量解释，并且可以通过模型来定量描述，其中每个物种都用等效的硬球表示，其大小与实际蛋白质分子的大小大致相等。研究还发现，在Mg的存在下，给定总浓度的FtsZ自缔合程度随着Hb或BSA浓度的增加而显著增加，其定量方式可以用排除体积理论来描述，在该模型中，FtsZ的低聚物用球形圆柱体表示，其固定半径近似等于单体FtsZ的半径，长度由质量守恒决定。通过测量微管蛋白溶液的波长依赖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，研究了控制大阵列微管蛋白组装的速率和平衡。显然，“惰性”添加剂的加入大大加速和增加了阵列形成的平衡程度，定性地符合排除体积理论的预测。装配过程的进一步实验和定量模型的制定正在进行中。在dna结合蛋白CAP和含有CAP特异性序列的42碱基对寡核苷酸之间形成特异性复合物的过程，已通过示踪沉降平衡在高浓度“惰性”蛋白存在和缺乏的情况下进行了研究，试图辨别惰性蛋白对CAP对寡核苷酸亲和力的影响。目前正在为这一过程拟订数量模型。采用时效浊度法和时效造粒法研究了葡聚糖（一种惰性水溶性聚合物）对载脂蛋白CII形成淀粉样纤维速率的影响。研究发现，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多达8倍，其方式可通过简单的排除体积模型定量描述。通过吸光度和近、远紫外圆二色性的温度依赖性研究了葡聚糖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，发现半变性温度有一个小而显著的升高。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.