Measurement of biomolecular interactions and association via light scattering

通过光散射测量生物分子相互作用和缔合

• 批准号: 8553392
• 负责人: Allen P Minton
• 金额: $ 31.5万
• 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/8553392
• 关键词: Accounting; Data; Dependence; Diffusion; Exhibits; Fluorescence; Guanine Nucleotides; Guanosine Triphosphate; Laboratories; Light; Measurement; Measures; Modeling; Molecular Weight; Monitor; Ovalbumin; Ovomucin; Phase Transition; Potassium; Proteins; Reporting; Solutions; Soybeans; Spectrum Analysis; Sucrose; Techniques; Thermodynamics; Trypsin Inhibitors; analog; instrumentation; light scattering; macromolecule; particle; protein function; sedimentation velocity; small molecule; solute; stoichiometry

1. The composition dependence of the light scattering intensity of mixtures of each of four proteins, BSA, ovalbumin, ovomucoid, and soybean trypsin inhibitor, at concentrations up to 2 mg/ml, and a small molecule, sucrose, at concentrations of up to 200 g/l was measured using a technique and instrumentation developed in this laboratory and described in previous reports. The results were analyzed using formalism developed in this laboratory appropriate for binary mixtures of non-associating solutes exhibiting repulsive self- and hetero-interaction at high concentration. The results could be quantitatively accounted for by a semiempirical model, according to which the protein and sucrose are represented as equivalent spherical particles, the effective volume of which depends upon self- and hetero-interactions. The results indicate that sucrose interacts with itself and with proteins through a combination of steric repulsion and weak attraction that partially compensates for the steric repulsion. The magnitude of the attractive interaction between sucrose and protein varies between proteins, and is greatest for soybean trypsin inhibitor and smallest for ovalbumin (D. Wu). 2. The self-association of the bacterial septation protein FtsZ in the presence of GTP or a GTP analogue, GMPCPP, and 500 mM potassium was studied as a function of protein and Mg concentration. Data obtained from several biophysical measurements, including static and dynamic light scattering, sedimentation velocity, and fluorescence correlation spectroscopy reveal that with increasing concentration, the protein undergoes a concerted formation, akin to a second-order phase transition to a narrow distribution of high molecular weight oligomers with a stoichiometry of ca 80 (GTP) or 120 (GMPCPP). Above the transition concentration, the amount, but not the size, of oligomer increases, while that of low molecular weight species remains roughly constant (Monterroso, Rivas). All of the data obtained from the several types of measurements may be accounted for semiquantitatively in the context of a model, according to which the stable oligomer is a cyclic species, the size of which reflects the tendency of the protein fibrils to flex or curve in solution in the presence of different concentrations of Mg and the particular guanine nucleotide present (B. Monterroso, G. Rivas, CIB).

1.利用本实验室开发的一种技术和仪器，测量了四种蛋白质(牛血清白蛋白、卵清蛋白、类卵粘蛋白和大豆胰蛋白酶抑制剂)和小分子蔗糖(浓度高达200g/L)的混合物的光散射强度随组成的变化。用本实验室发展的适用于非缔合溶质的二元混合物的公式对结果进行了分析，这些混合物在高浓度下表现出排斥的自作用和异作用。这一结果可以用半经验模型来定量解释，根据该模型，蛋白质和蔗糖被表示为等效的球形颗粒，其有效体积取决于自体和异体相互作用。结果表明，蔗糖通过空间排斥力和弱吸引的组合与自身和蛋白质相互作用，从而部分补偿空间排斥力。蔗糖与蛋白质之间吸引作用的大小因蛋白质的不同而不同，其中大豆胰酶抑制剂的吸引作用最大，卵清蛋白的吸引作用最小。 2.研究了细菌分离蛋白FtsZ在GTP或GTP类似物GMPCPP和500 mM钾存在下的自结合作用及其与蛋白质和镁浓度的关系。包括静态和动态光散射、沉降速度和荧光相关光谱在内的几种生物物理测量数据表明，随着浓度的增加，蛋白质经历了协同形成，类似于二级相变，即化学计量比约为80(GTP)或120(GMPCPP)的高分子量低聚物的窄分布。在转变浓度以上，齐聚物的数量增加，但大小不变，而低分子量物种的数量大致保持不变(蒙特罗索，里瓦斯)。从几种类型的测量获得的所有数据可以在一个模型的背景下半定量地解释，根据该模型，稳定的低聚物是环状物种，其大小反映了在存在不同浓度的镁和存在的特定鸟嘌呤核苷酸的情况下蛋白质原纤维在溶液中弯曲或弯曲的趋势(B.蒙特罗索，G.Rivas，CIB)。