Fluorescence quenching dynamics of single antigen-antibody pairs under an applied load

 

Will Heinz, Peter Yim, Lori Goldner

 

Optical Technology Division, Physics Laboratory

 

 

Interactions between individual molecules depend on variables such as separation distance, orientation, conformation, and local environment. Atomic force microscope (AFM) measurements on ligand-receptor pairs have shown the unbinding force depends on the loading rate and enthalpy of binding; however, these measurements do not detect the orientational and conformational changes of non-load bearing domains of the molecules and are also limited by incomplete descriptions of the number of interacting pairs contributing. Single-molecule fluorescence spectroscopy (SMFS) techniques are sensitive to molecular conformation, orientation, and dynamics. Combining an AFM with SMFS measurements can more completely describe the interaction between single molecules. Here we present a combined AFM and scanning confocal microscope capable of single molecule detection and manipulation. Using a simple system, antibodies directed against a fluorophore, we investigate the coupled intermolecular interactions and fluorescence dynamics of a ligand-receptor pair during forced binding/unbinding. We present simultaneous SMFS measurements on the AFM forced-unbinding of single antigen-antibody pairs in aqueous buffer and examine correlations between fluorescence intensity, separation distance, applied load, and binding state.