Interfacial molecular binding events measured by single molecule imaging

Molecular interactions are critical for every aspect of biological function. These interactions govern cell signaling, life-cycle regulation, biosynthetic pathways, the immunological response, control of gene expression, and vesicle-membrane fusion. Measuring the affinities of these interactions and their kinetics of binding and unbinding is therefore critical to understanding biology at the molecular level. In this work total internal reflection fluorescence (TIRF) microscopy was used to detect single molecule molecular interactions. The density of surface-immobilized ligands or binding sites is an important issue for the development of single-molecule detection methods. Control of the binding site density of reactive ligands on surfaces was accomplished by diluting surface amine groups in self-assembled and cross-linked monolayers on glass prepared from solutions containing very low concentrations of an amine terminated triethoxy-silane (either 3-aminoproyltriethoxy-silane or amine-PEG5000 triethoxy-silane) and much higher concentrations of 2-cyanoethyltriethoxy-silane. The surface amine sites are suitable for attaching labels and ligands by reaction with varying reagents. After characterization of the modified surfaces using single-molecule imaging of surface immobilized dyes and ligands, the surfaces were tested to determine the ability to observe single molecule binding kinetics. A single-molecule fluorescence method was developed for measuring the kinetics and affinity constant for the binding of neutravidin, a deglycosylated variant of avidin, to surface-immobilized biotin. TIRF microscopy was used to image the binding kinetics. Having successfully imaged the binding kinetics of the long lived neutravidin-biotin complex the ability to observe kinetics of a protein-protein complex were examined. The protein complex chosen for these experiments was the IgG antibody anti-syntaxin binding to immobilized syntaxin. Mammalian and Caenorhabditis elegans (worm) variants of syntaxin were harvested from E. coli and immobilized to a cysteine modified glass surfaces using a thioester linker. Fluorescence imaging of the protein interactions was accomplished by labeling anti-syntaxin with Cy3B. Binding experiments of the syntaxin-anti-syntaxin complex were at a bovine serum album passivated glass coverslip and total internal reflection fluorescence microscopy.