Single molecule level studies can lay the foundation towards understanding the microscopic nature of interaction forces between ligands and enzymes that in turn strongly influence majority of biological processes. Study of interactions between individual molecules is not accessible from experiments utilizing a large ensemble of molecules. Such information is important since the averaged properties may not truly represent the single molecular responses. This study utilizes high spatial and force resolution provided by the Atomic Force Microscopy (AFM) under physiological conditions to quantify the force-distance information of enzyme-ligand interaction. Different immobilization techniques were utilized to chemically bind the enzyme to the surface and ligand molecules to the AFM tip and then results were quantitatively compared to improve the accuracy and reproducibility of the measurements. Measurements of forces required to rupture enzyme-ligand complex were carried out on directly immobilized enzymatic monolayer and compared with enzymes bound to surface through a rigid double stranded (ds) DNA spacers. Ligand molecules were attached to the tip via either flexible polyethylene glycol or rigid dsDNA linkers. This 123-base pair containing 41 nm long rigid dsDNA linker was proved to withstand repetitive force measurements of enzyme-ligand rupturing. The findings of this study indicate that new single molecule approach for measuring enzyme-ligand interactions based on rigid dsDNA linker on both tip and surface afford highly specific and accurate force measurements.

Single molecule level studies of enzyme-ligand interactions using molecular recognition atomic force spectroscopy

256th National Meeting (2018)

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