Measurement of biomolecular association via static and dynamic light scattering

通过静态和动态光散射测量生物分子缔合

• 批准号: 8148691
• 负责人: Allen P Minton
• 金额: $ 30.67万
• 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/8148691
• 关键词: Measurement; light scattering

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).

1. 我们同时收集了非相互作用和相互作用蛋白质混合物的成分依赖性静态和动态光散射数据，并对这两个量的成分依赖性进行了全局分析。 与预期相反，添加动态光散射数据并没有提高仅使用静态光散射数据可获得的表征的分辨率。 这项工作的结果正在撰写中以供出版。 （B·蒙特罗索） 2. 通过测量静态光散射的浓度依赖性（A. Attri），我们表征了无锌胰岛素在广泛 pH 值范围内的自缔合平衡。 在 pH 1.6 时，胰岛素在醋酸盐缓冲液中是非缔合单体，在 HCl 中是非常弱的自缔合二聚体。 pH 值在 3 到 8 之间时，散射的浓度依赖性可以通过简单的等脉不定缔合方案定量地解释。 在pH 10时，散射的浓度依赖性通过改进的等阻方案定量地解释，其中单体向单体添加的平衡缔合常数比单体向所有高级低聚物添加的平衡缔合常数小约五倍。 这项工作的报告已发表（A. Attri、C. Fernandez）。 3. 我们已经表征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，在研究的浓度范围内（0.3 - 5 mg/ml），胰岛素表现为六聚体，经历等脉无限自缔合。 对动态光散射浓度依赖性的分析证实了这一解释。 这项工作的报告已发表（A. Attri、C. Fernandez）。 4. 我们表征了 tau 蛋白与肝素（一种具有高负电荷密度的硫酸化多糖）的 7K 分子量级分之间的相互作用。 散射强度的成分依赖性可以通过简单的 1:1 异质缔合很好地描述。 这是成分梯度-静态光散射首次应用于两种不同类型大分子之间的缔合。 这项工作的报告已发表（C. Fernandez、Y. Liang）。 5. 我们测量了在不同浓度的 Mg++ 离子存在下，含有 GTP 和 GTP 再生系统或 GPCPP（一种可缓慢水解的 GTP 类似物）的 FtsZ 溶液中光散射强度对蛋白质浓度的依赖性。 对观察到的依赖性的最简单解释是，FtsZ-GTP 或 FtsZ-GPCPP 的自缔合最初根据不定的等脉方案通过线性增长进行，然后协同形成稳定的低聚物或尺寸为 30 的窄尺寸分布的低聚物-40 个被认为是循环的亚基。 这项工作正在准备出版（G. Rivas）。