Elucidating the structure of proteins, nucleic acids, and assemblies of these macromolecules provides information critical to understanding and ultimately enabling the control of biological function. Atomic force microscopy (AFM) is a powerful technique that has been utilized to probe the structure and dynamics of biological systems, and thus can advance significantly knowledge of biological function. The level of information in AFM images depends critically on the size, shape and terminal functionality of the tips used for imaging. Commercial tips have exhibited impressive resolution on packed molecular arrays but lower resolution on isolated proteins, and can show significant tip-to-tip variations in resolution. To overcome limitations of present tips and better exploit the potential of AFM, the present study will focus on the development and application of carbon nanotube probes. Carbon nanotubes have several features that make them ideal for structural biological, including high aspect ratios for imaging deep and narrow features and potential resolution better than 0.5 nanometers. Moreover, the well-defined molecular structure of nanotubes should enable the synthesis of identical size and resolution tips, and the modification of nanotube ends for imaging with chemical sensitivity. The overall aims of this project are to develop the methodologies needed to prepare carbon nanotube tips with reproducible ultrahigh structural resolution, to develop approaches for modifying nanotubes ends for functional imaging, and to exploit carbon nanotube tips to elucidate the mechanism of chromatin remodeling by SWI/SNF and other complexes. Metal-catalyzed chemical vapor deposition will be used to synthesize and thereby directly control the carbon nanotube tips. Electron microscopy imaging of nanotube tips and AFM imaging of standards and protein model systems with the same tips will be used to define the relationships between synthesis, structure and resolution. Chemical reactions will be used to localize probe species, including basic organic functional groups and more complex ligands, at the ends of nanotube tips. The resolution of the modified probes in mapping chemically-distinct residues and binding sites will be defined using monolayer, bilayer and protein systems. Carbon nanotube tips will be used to determine the structures of the products produced by ATP-dependent SWI/SNF remodeling of mononucleosomes and polynucleosomes, to study the structure(s) of the SWI/SNF complex, to analyze the role that higher order chromatin structure has on remodeling, and to study remodeling by nucleosome remodeling deacetylase complex.
