PROTEIN MEDIATED MOLECULAR ADHESION--AFM STUDIES

蛋白质介导的分子粘附--AFM 研究

• 批准号: 2701813
• 负责人: VINCENT T MOY
• 金额: $ 9.86万
• 依托单位: UNIVERSITY OF MIAMI SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-05-01 至 2002-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2701813
• 关键词: atomic force microscopy; binding proteins; biotin; calorimetry; cell adhesion; chemical association; chemical bond; fluoresceins; gel filtration chromatography; intermolecular interaction; monoclonal antibody; scintillation counter; site directed mutagenesis; thermodynamics

Protein-mediated molecular adhesion: AFM studies. Numerous cellular processes including cell adhesion and cell migration are mediated by the transient interaction of adhesion molecules. Often, as in the case cell migration, there is a delicate balance between cell attachment and cell de-attachment. The strength of cell attachment is determined by the number of intermolecular bonds formed between membrane- bound receptors and their ligands on the opposing surface and the de- attachment force of the individual bonds. Whereas the number of bonds formed at the interface is closely related to the association constant of the protein-ligand interaction, the rupture force of these bonds is poorly understood and is the main focus of this proposal. The long-term objective of this proposed research is to achieve a fundamental understanding of the mechanisms involved in molecular adhesion. The current proposal examines the intermolecular forces of protein-ligand interaction in two model systems, the streptavidin-biotin pair and an antibody-antigen pair. In general, protein-ligand bonds are noncovalent and will spontaneously break by thermal agitation given sufficient time. The dissociation lifetime of the protein-ligand bond is accelerated and can be studied by applying an external force across the bond with an Atomic Force Microscope (AFM). Comprehensive measurements of force-life time relationships derived from AFM-induced separation of the streptavidin- biotin bond will be used to reveal the dissociation pathway and possible intermediate states of the complex. AFM measurements of mutagenized streptavidin will be used to identify key amino acid determinants responsible for adhesion to biotin. In contrast to the resilience of the streptavidin-biotin interaction, the binding of fluorescein to 4-4-20, an anti-fluorescyl antibody, can be thermodynamically manipulated by small changes in temperature, pH, and solvent system. The effects of these perturbations on the dissociation constant and binding enthalpy will be studied and correlated to force measurements to determine the reaction pathway of 4-4-20:fluorescein dissociation beyond the point of initial bond rupture. Together, these experiments will contribute to establishing a conceptual framework for understanding protein-mediated molecular adhesion.

蛋白质介导的分子粘附：AFM研究。 包括细胞粘附和细胞迁移在内的许多细胞过程是 由粘附分子的瞬时相互作用介导。 通常，如 案例细胞迁移，细胞之间存在微妙的平衡 附着和细胞脱位。 细胞附着的强度是 由膜之间形成的分子间键的数量确定 结合受体及其配体在相对表面和de- 单个键的依恋力。 而债券的数量 在接口处形成的与 蛋白质 - 配体相互作用，这些键的破裂力很差 理解，是该提议的主要重点。 这项拟议研究的长期目标是实现 对分子粘附涉及的机制的基本理解。 当前的提案检查了蛋白质配体的分子间力 在两个模型系统中的相互作用，链霉亲和素 - 生物素对和一个 抗体 - 抗原对。 通常，蛋白质 - 配体键是非共价的 并在足够的时间下自发地因热搅拌而自发破裂。 蛋白质键的解离寿命是加速的，可以 通过用原子施加外力来研究 力显微镜（AFM）。 力量寿命时间的全面测量 从AFM诱导的链霉亲和素分离得出的关系 生物素键将用于揭示解离途径并可能 综合体的中间状态。 AFM测量诱变 链霉亲和素将用于识别关键氨基酸决定因素 负责对生物素的粘附。 与 链霉亲和素生物素相互作用，荧光素与4-4-20的结合，A 抗氟乙烯抗体，可以通过小型小学操纵 温度，pH和溶剂系统的变化。 这些影响 解离常数和结合焓的扰动将是 研究并与强制测量相关以确定反应 4-4-20的途径：超出初始键点的荧光素解离 破裂。 这些实验共同有助于建立 理解蛋白质介导的分子粘附的概念框架。