Measurement of biomolecular association via static and dynamic light scattering

1.我们同时收集了非相互作用蛋白质和相互作用蛋白质混合物的与组成相关的静态和动态光散射数据，并对这两个量的组成依赖关系进行了全局分析。与预期相反，动态光散射数据的添加并没有提高仅使用静态光散射数据所能获得的表征的分辨率。这项工作的成果正在撰写中，准备出版。(B.蒙特罗索) 2.通过测量静态光散射的浓度依赖关系(A.Attri)，我们表征了无锌胰岛素在广泛的pH值范围内的自缔合平衡。在pH为1.6时，胰岛素在醋酸盐缓冲溶液中为非缔合单体，在盐酸中为弱自缔合二聚体。在pH值为3-8时，散射的浓度依赖关系可用一个简单的等键不定缔合方案来定量解释。在pH为10时，散射的浓度依赖关系可用改进的等键吸附方案来定量解释，其中单体与单体加成的平衡缔合常数约为单体与所有较高齐聚物加成的平衡缔合常数的五分之一。这项工作的报告已经发表(A.Attri，C.Fernandez)。 3.研究了锌存在下胰岛素的自缔合平衡。静态光散射的浓度依赖性分析表明，在所研究的浓度范围内(0.3-5 mg/ml)，胰岛素表现为经历等键不确定自缔合的六角体。对动态光散射的浓度依赖关系的分析证实了这一解释。这项工作的报告已经发表(A.Attri，C.Fernandez)。 4.我们研究了tau蛋白与7K分子量肝素的相互作用，肝素是一种具有高负电荷密度的硫化多糖。散射强度与组份的关系可以用简单的1：1异质缔合来描述。这是首次将组成梯度静态光散射应用于两种不同类型的大分子之间的缔合。这项工作的报告已经发表(C.Fernandez，Y.Leung)。 5.我们测量了在不同浓度的镁离子存在下，含有GTP和GPCPP的FtsZ溶液中的光散射强度与蛋白质浓度的关系。对观察到的依赖性的最简单解释是，FtsZ-GTP或FtsZ-GPCPP的自缔合最初是根据不确定的等键方案通过线性增长进行的，随后合作形成稳定的低聚物或被认为是环状的30-40亚基的窄尺寸分布的低聚物。这部作品正准备出版(G·里瓦斯)。

1. We have simultaneously collected composition-dependent static and dynamic light scattering data on mixtures of non-interacting and interacting proteins, and developed a global analysis of the composition-dependence of both quantities. Contrary to expectation, addition of dynamic light scattering data did not enhance the resolution of characterization obtainable using static light scattering data only. Results of this work are being written up for publication. (B. Monterroso) 2. We have characterized the self-association equilibria of zinc-free insulin over a wide range of pH values, via measurement of the concentration dependence of static light scattering (A. Attri). At pH 1.6, insulin is a non-associating monomer in acetate buffer and a very weakly self-associating dimer in HCl. At pH values between 3 and 8, the concentration dependence of scattering is quantitatively accounted for by a simple isodesmic indefinite association scheme. At pH 10, the concentration dependence of scattering is quantitatively accounted for by a modified isodesmic scheme in which the equilibrium association constant for addition of monomer to monomer is about five times smaller than the equilibrium association constant for addition of monomer to all higher oligomers. A report of this work has been published (A. Attri, C. Fernandez). 3. We have characterized the self-association equilibria of insulin in the presence of zinc. Analysis of the concentration dependence of static light scattering indicates that over the concentration range studied (0.3 - 5 mg/ml), insulin behaves as a hexamer undergoing isodesmic indefinite self-association. Analysis of the concentration dependence of the dynamic light scattering confirms this interpretation. A report of this work has been published (A. Attri, C. Fernandez). 4. We have characterized the interaction between tau protein and a 7K molecular weight fraction of heparin, a sulfated polysaccharide with high negative charge density. The composition dependence of the scattering intensity is well described by a simple 1:1 heteroassociation. This is the first application of composition gradient - static light scattering to the association between two different types of macromolecule. A report of this work has been published (C. Fernandez, Y. Liang). 5. We have measured the dependence of the intensity of light scattering upon protein concentration in solutions of FtsZ containing either GTP an a GTP-regenerating system or GPCPP, a slowly hydrolyzable GTP analog, in the presence of varying concentrations of Mg++ ion. The simplest interpretation of the observed dependence is that self-association of either FtsZ-GTP or FtsZ-GPCPP proceeds initially by linear growtn according to an indefinite isodesmic scheme, followed by cooperative formation of a stable oligomer or narrow size distribution of oligomers of size 30-40 subunits that are thought to be cyclic. This work is being written up for publication (G. Rivas).