Fast and Angström-resolution AFM to visualise conformational change in biomolecules

Atomic Force Microscopy (AFM) basically acts as a miniature blind man's stick ('cantilever') following the contours of a sample surface, and line by line reconstructing a three-dimensional representation of the surface topography. This line-by-line scanning is a fundamental difference from other, more common microscopy techniques and a main reason why it generally takes minutes to complete a single image. AFM is unique in combining sub-molecular resolution imaging with the ability to operate in liquids. For high-resolution imaging of biological samples, molecules are generally adsorbed on a hard surface, which is the only compromise compared to physiological conditions. Membrane proteins are samples of particular interest, since they represent more than 50% of modern drug targets and therefore are of major pharmaceutical importance. Their function as molecular nanomachines is determined by Angstrom-sized structural ('conformational') changes occurring at millisecond time scales. For applications in future healthcare and for basic scientific understanding, the crucial question is how molecular structure and changes in this structure relate to the biological function of membrane proteins. This project combines high-resolution AFM techniques (that have yielded atomic resolution!) with fast scanning, to obtain images of membrane proteins with Angstrom spatial and millisecond temporal resolution. This will enable us to visualise conformational changes in real time and observe biomolecules at work. This will be demonstrated on bacteriorhodopsin, a light-driven molecular machine that pumps protons through the cell membrane.

The function of biomolecular machinery is determined by Angstrom-sized motion at millisecond time scales. This proposal outlines a development that will combine Angstrom-resolution atomic force microscopy (AFM) with fast scanning technology. The aim is real-time visualisation of Angstrom-sized conformational changes in membrane proteins under physiological conditions. This will be demonstrated by measuring the conformational changes that determine the direction of proton pumping in bacteriorhodopsin. The technique will be readily applied to the study of other biomolecular machines. First attempts to develop fast AFM date back to at least a decade ago. Several separate components necessary for fast and high-resolution AFM, however, have only been completed recently, and are yet to be integrated within an approach that yields Angstrom resolution at conventional scan speeds to start with. In particular, the highest-resolution operation modes at conventional speeds --- contact mode and FM-AFM, with accurate control of the tip-sample distance --- are yet to be implemented at high scan speeds. This will be the main instrumental innovation of this work. In addition, the scan-line triggered illumination will yield a much higher effective time resolution than can be achieved by monitoring molecules frame-by-frame only. Success in this direction depends on miniaturisation of cantilevers and on the ability to measure their deflections with highest sensitivity, such as can be achieved by the applicant's home-built deflection sensor.