Mechanical Mapping of Single Proteins at Subnanometer Resolution using AFM

Abstract

The structure and function of proteins are determined by their mechanical stability. The temperature- or B-factors obtained from crystallographic data provide an indirect measure of the order and stability of the protein, together with structural information. However, B-factors from crystallography analysis may be related to the protein packing in the 3D-crystal and are not quantitative. A direct quantification of the mechanical stability of proteins based on atomic force microscopy (AFM) consists in mechanically unfolding individual proteins by pulling from two points. However, its correlation with protein structure requires additional measurements. In this work we apply a novel AFM imaging mode based on force spectroscopy (PeakForce) to simultaneously acquire structural and mechanical information of membrane proteins. We used the well-known protein bacteriorhodopsin from Halobaterium salinarum as a model system, obtaining topographical and stiffness maps with subnanometer resolution. The characteristic trimeric organization of the trigonally packed (a = b = 62.5A; γ= 120°) bacteriorhodopsin was clearly visible with inter-monomer distances of 3.2 nm. Overlay of stiffness and topography maps allowed the investigation of nanomechanical properties at the single molecule level providing a map of the structural stability of bacteriorhodopsin.View Large Image | View Hi-Res Image | Download PowerPoint Slide