PROTEIN MEDIATED MOLECULAR ADHESION--AFM STUDIES

蛋白质介导的分子粘附--AFM 研究

• 批准号: 2024166
• 负责人: VINCENT T MOY
• 金额: $ 10.89万
• 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/2024166
• 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：原子力显微镜研究。 许多细胞过程，包括细胞粘附和细胞迁移， 由粘附分子的瞬时相互作用介导。 通常，如 在细胞迁移情况下，细胞之间存在微妙的平衡 附着和细胞去附着。 细胞附着的强度是 这取决于膜与膜之间形成的分子间键的数量， 结合受体及其配体在相对表面上， 单个键的附着力。 而债券的数量 在界面处形成的缔合常数与 蛋白质-配体相互作用，这些键的断裂力很差 这是理解的，也是这项建议的重点。 这项拟议研究的长期目标是实现 对分子粘附机制的基本理解。 目前的建议是研究蛋白质-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 抗体-抗原对 一般来说，蛋白质-配体键是非共价的 并且在足够的时间内通过热搅拌将自发地破裂。 蛋白质-配体键的解离寿命被加速， 通过在键上施加外力来研究， 力显微镜（AFM）。 力-寿命时间的综合测量 来自AFM诱导的链霉亲和素分离的关系- 生物素键将用于揭示解离途径和可能的 复杂的中间状态。 原子力显微镜测量诱变的 链霉亲和素将用于鉴定关键氨基酸决定簇 负责与生物素的粘附。 相比之下， 链霉亲和素-生物素相互作用，荧光素与4-4-20的结合， 抗荧光抗体，可以通过小的 温度、pH和溶剂系统的变化。 成效为何 对离解常数和结合焓的扰动将是 研究并与力测量相关联，以确定反应 4-4-20途径：荧光素解离超过初始键合点 破裂 总之，这些实验将有助于建立一个 理解蛋白质介导的分子粘附的概念框架。