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.
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