PROTEIN MEDIATED MOLECULAR ADHESION--AFM STUDIES

蛋白质介导的分子黏附：原子力显微镜研究。 包括细胞黏附和细胞迁移在内的许多细胞过程 由黏附分子的瞬时相互作用所调节。通常，如在 在细胞迁移的情况下，细胞之间存在微妙的平衡 贴壁和细胞脱附。细胞附着的强度是 由膜之间形成的分子间键的数量决定- 结合在相对表面上的受体及其配体和脱氢酶 单个键的附着力。鉴于债券的数量 在界面形成与缔合常数有密切关系 蛋白质与配体的相互作用，这些键的断裂力很差。 理解这一点，也是这项建议的主要重点。 这项拟议研究的长期目标是实现 对分子粘合的机理有基本的了解。 目前的方案研究了蛋白质-配基的分子间力。 链霉亲和素-生物素对和AN两个模型体系的相互作用 抗体-抗原对。一般来说，蛋白质-配位键是非共价的。 如果有足够的时间，热搅拌会自发破裂。 蛋白质-配位键的解离寿命被加速，并且可以 通过在原子键上施加外力来研究 力显微镜(AFM)。力-寿命的综合测量 从AFM诱导的链霉亲和素分离得到的关系- 生物素键将被用来揭示解离途径和可能 复合体的中间状态。诱变剂的原子力显微镜测量 链霉亲和素将用于识别关键的氨基酸决定因素 负责与生物素的粘连。与之形成鲜明对比的是， 链霉亲和素-生物素相互作用，荧光素与4-4-20的结合，以及 抗荧光素抗体，可由小分子热力学操纵 温度、pH和溶剂体系的变化。这些因素的影响 对离解常数和结合热的微扰将是 研究并关联测力以确定反应 4-4-20途径：荧光素在起始键点以外的解离 破裂。总之，这些实验将有助于建立一种 理解蛋白质介导的分子黏附的概念框架。

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.