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: 7967202
• 负责人: Allen P Minton
• 金额: $ 19.12万
• 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/7967202
• 关键词: Accounting; Acetates; Aliquot; Annual Reports; Biological; Buffers; Cells; Charge; Concentration measurement; Data; Dependence; Diffusion; Equilibrium; Goals; Heparin; Inorganic Sulfates; Insulin; Kinetics; Laboratories; Light; Measurement; Measures; Modeling; Molecular Weight; Monitor; Nature; Polysaccharides; Process; Property; Proteins; Publications; Radial; Reaction; Resolution; Scheme; Solutions; Techniques; Thermodynamics; Time; Unspecified or Sulfate Ion Sulfates; Weight; Work; base; density; design; dimer; expectation; instrument; light scattering; macromolecule; monomer; protein aggregate; protein aggregation; research study; tau Proteins

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 have been submitted for publication. (B. Monterroso) 2. We have constructed two different instruments designed to simultaneously measure time-dependent static and dynamic light scattering of proteins undergoing aggregation. The first instrument is a cuvette-based instrument that will measure 90o static and dynamic light scattering. The second is an instrument that will automatically extract aliquots of aggregating protein from a reaction vessel and introduce it into a flow cell for measurement of static light scattering at multiple angles and dynamic scattering at a single angle. Our goal is to obtain, in a single experiment, the time dependence of weight-average molecular weight, z-average radius of gyration, and the intensity-weighted distribution of diffusion coefficients. Modeling of the three quantities should provide a high resolution picture of the kinetic scheme of aggregation. (A. Attri, C. Fernandez) 3. We have characterized the self-association equilibria of 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. Attri) 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.

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静态和动态光散射数据，并对这两个量的组成依赖性进行了全局分析。与预期相反，加入动态光散射数据并没有提高仅使用静态光散射数据获得的表征分辨率。这项工作的结果已提交出版。(b .参与者蒙特罗索这样)