Biophysical Characterization Of Macromolecules

大分子的生物物理表征

• 批准号: 7593812
• 负责人: PETER SCHUCK
• 金额: $ 11.1万
• 依托单位: NATIONAL INSTITUTE OF BIOMEDICAL IMAGING AND BIOENGINEERING
• 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/7593812
• 关键词: 76-kDa SH2 domain-containing leukocyte protein; Adaptor Signaling Protein; Affinity; Anthrax disease; Antibodies; Antigens; Area; Binding; Biological; Biosensing Techniques; Biosensor; Calorimetry; Circular Dichroism Spectroscopy; Collaborations; Complex; Computational Technique; Coupled; Cytomegalovirus; Dengue Virus; Differential Scanning Calorimetry; Diffusion; Glutamate Receptor; Glycoproteins; Goals; Interferon Alfa-2c; Interferon Receptor; Interferons; Japanese encephalitis virus; Kinetics; LCP2 gene; Laboratories; Ligands; Measures; Methodology; Methods; Phosphorylation; Proteins; Rate; Research; Resolution; Role; Signal Transduction; Solutions; Staging; Standards of Weights and Measures; Surface Plasmon Resonance; System; T-Cell Activation; Techniques; Titrations; Vaccinia virus; Variant; analytical ultracentrifugation; chemical reaction; crosslink; experience; human WNT2 protein; in vivo; light scattering; macromolecule; migration; new technology; novel; particle; receptor binding; sedimentation coefficient; sedimentation velocity; stoichiometry; tool

A major application of the computational affinity- and kinetic rate distribution analysis previously developed for enhanced surface plasmon resonance biosensing was the characterization of antibodies against antigens derived from Japanese encephalitis virus, Dengue virus, vaccinia virus, and different anthrax proteins, all in collaboration with LID/NIAID. For most targets, several antibodies were characterized and their affinity compared in detail. A second application area of biosensing, in conjunction with analytical ultracentrifugation, was the study of the interferon receptor IFNAR-2 interaction with interferons alpha-2c and CM3, in collaboration with DIR/NIAID. Here, qualitative biosensor studies on the cooperativity of the two ligands and the discovery of the self-association of interferon alpha-2c added to the novel finding of the mutual influence of the interferons in their receptor binding. As an application of our techniques for the study of multi-protein interactions in solution by multi-signal sedimentation velocity analytical ultracentrifugation and by isothermal titration calorimetry, we have embarked with LCMB/NCI on the collaborative characterization of adaptor proteins essential for T-cell activation. Previously, collaborative studies indicated the role of multi-valent adaptor protein cross-linking in the formation of signaling particles in vivo. Using similar methodology, we have examined the ternary system of interactions between an SLP-76 fragment in different phosphorylation states, Nck, and Vav. The results suggest the formation of triple protein complexes of various stoichiometries. Analytical ultracentrifugation provides unique information on protein self-association and hetero-association. We have previously developed techniques for the computational deconvolution of diffusion to arrive at high-resolution sedimentation coefficient distributions, and have shown theoretically how these can be interpreted quantitatively in the framework of coupled migration and chemical reaction, representing a robust method for measuring macromolecular binding constants. This tool was applied in collaboration with LI/NIAID to the characterization of different cytomegalovirus glycoproteins (putative immunoevasins) and interaction with a NKG2D ligand. In a different context, this approach was used to study the role of self-association of glutamate receptor variants. Further, several additional collaborative projects with different IRP laboratories on the characterization of proteins are in a pilot stage, or were conducted using standard biophysical methodology.

一个主要的应用程序的计算亲和力和动力学速率分布分析先前开发的增强表面等离子体共振生物传感是表征抗体的抗原来自日本脑炎病毒，登革热病毒，牛痘病毒，和不同的炭疽蛋白，所有在LID/NIAID合作。 对于大多数靶标，表征了几种抗体，并详细比较了它们的亲和力。 生物传感与分析超离心相结合的第二个应用领域是与生物技术研究所/NIAID合作研究干扰素受体IFNAR-2与干扰素α-2c和CM 3的相互作用。 在这里，定性的生物传感器研究的协同性的两个配体和发现的自关联的干扰素α-2c添加到新的发现的相互影响的干扰素在其受体结合。 作为我们的技术的应用，在溶液中的多蛋白质相互作用的研究，通过多信号沉降速度分析超离心和等温滴定量热法，我们已经开始与LCMB/NCI的T细胞活化所必需的衔接蛋白的协作表征。 此前，合作研究表明多价衔接蛋白交联在体内信号颗粒形成中的作用。 使用类似的方法，我们研究了三元系统之间的相互作用的SLP-76片段在不同的磷酸化状态，Nck，和Vav。 结果表明，形成的三重蛋白质复合物的各种化学计量。 分析超电泳提供了关于蛋白质自缔合和异缔合的独特信息。 我们以前已经开发出的技术，扩散的计算去卷积，以达到高分辨率的沉降系数分布，并从理论上表明如何这些可以被定量解释的框架内的耦合迁移和化学反应，代表一个强大的方法测量大分子结合常数。 该工具与LI/NIAID合作应用于不同巨细胞病毒糖蛋白（推定免疫evasins）的表征以及与NKG 2D配体的相互作用。 在不同的背景下，这种方法被用来研究谷氨酸受体变体的自缔合作用。 此外，与不同的IRP实验室就蛋白质特性进行的其他几个合作项目正处于试验阶段，或使用标准生物物理方法进行。