Conformational changes of surface immobilized proteins studied by combined Atomic Force Microscopy and Fluorescence Spectroscopy

Atomic Force Microscopy (AFM) and Fluorescence Spectroscopy techniques have provided unique methods for characterizing conformational changes in proteins. Here we are using a technique called nanografting to immobilize proteins at well defined locations on atomically flat surfaces. In nanografting the AFM tip is used to shave alkanethiol molecules from a prescribed patch on a surface coated with an alkanethiol monolayer. Thiol-linked proteins in the surrounding solution are then able to self assemble on the newly exposed surface patch in a highly ordered array of the order of 100nm. Stable and meta-stable conformations of fluorescently tagged proteins and other molecules assembled in this manner can then be characterized using a combination of AFM and Fluorescent Resonance Energy Transfer (FRET). Due to the high spatial, temporal and force resolution provided by both AFM and FRET, a free energy landscape of the protein may be determined using this technique.