NONCOVALENT INTERMOLECULAR INTERACTIONS IN BIOCHEMISTRY

我们实验室最近发展的非理想示踪沉淀平衡技术被用来测量两种蛋白质中的任何一种(高达150g/L)对细菌细胞分裂蛋白FtsZ的热力学活性和/或缔合状态的影响。在无镁离子的情况下，FtsZ不自缔合，而在镁的存在下，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，在没有镁的情况下，FtsZ在Hb或BSA存在下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空间排斥的假设来定量解释，并可以用模型来定量描述，其中每个物种都由一个等效的硬球表示，其大小大致等于实际蛋白质分子的大小。研究还发现，在镁的存在下，对于给定的FtsZ总浓度，FtsZ的自缔合程度随着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，在该模型中，FtsZ的低聚物由球状柱状分子表示，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随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)随波长变化的浊度，研究了控制大阵列微管蛋白组装的速率和平衡。结果表明，“惰性”添加剂的加入极大地加速和提高了阵列形成的平衡程度，这与排除体积理论的预测是定性一致的。组装过程的进一步实验和定量模型的制定正在进行中。用示踪沉淀平衡法研究了DNA结合蛋白CAP与含有CAP特异序列的42碱基寡核苷酸在无惰性蛋白和高浓度“惰性”蛋白存在时形成的特异性复合体，以期弄清惰性蛋白对CAP与寡核苷酸亲和力的影响。这一过程的量化模型正在制定中。

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.