Force Spectroscopy of Biopolymers:Correlating Molecular Structure with Single Molecule Elasticity

Abstract

Single molecule mechanical testing is providing new insights into macromolecular behavior of complex native proteins including the energetics and dynamics of protein domain folding/unfolding and protein-ligand binding. The performance of atomic force microscopy (AFM) in single molecule mechanical measurements has distinct advantages over other force measuring devices. The very precise displacement measurements afforded protein molecules have relied largely on non-specific physisorption of the molecules to bare substrates by the AFM scanner enables examination of single molecules that are much smaller that those examined by other techniques (e.g. optical tweezers). Current AFM methods for investigation of single using the eponymous "smash and grab' technique (see FIG. 1). This involves bringing the tip into contact with the protein adsorbate followed by tip retraction at a controlled rate. However, the point of contact with the molecule and its orientation during loading is unknown. As a result, the observed length at release is related only to the point of contact and not to the structure of the macromolecule (see FIG. 2). Only the persistence length can be extracted from fits of either the freely jointed or worm-like chain models to the force-separation data.We envision a strategy for mechanical testing of proteins with well-defined attachments at the termini of the polypeptide chain to the tip and substrate surfaces (see FIG. 3). This strategy will provide a rapid and reliable method for analysis of the macromolecular properties of these biomolecules that is free of the artifacts and assumptions implicit in data acquisition with the "smash and grab" technique. We are examining the utility of molecular recognition between streptavidin and biotin to make contact at a known position on a single biomolecule. Biotinylated molecules are covalently immobilized onto a tip array whereas streptavidin is covalently attached to a tipless cantilever. This strategy enables replicate measurements on the same molecule. Force-separation curves obtained for tensile-loaded molecules are analyzed using both the freely-jointed and worm-like chain models. Our present efforts are devoted to correlating the mechanical properties with nucleotide sequence, assessing the impact of streptavidin unfolding during tensile loading of DNA, and extending this approach to elastin mimetic synthetic polypeptides.