The scanning tunneling microscope routinely provides atomic-resolution imaging and spectroscopic measurements of single molecules on surfaces. The primary obstacle preventing the full power of this capability from being applied to biological targets such as proteins is the limitation that, in general, electrons cannot tunnel through more than 2 nm of an insulating material. This restriction impacts entire classes of biologically interesting samples such as proteins (typically ~5 nm diameter) and lipid bilayers (~6 nm thickness). This proposal describes the development of instrumentation and methodologies expanding the sub- nanometer imaging capabilities of scanning tunneling microscopy (STM) to biologically important single molecules and biomolecule assemblies on surfaces, including proteins embedded in lipid membranes. In particular, this approach will allow direct observations and structural studies of G protein-coupled receptor (GPCR)-ligand complexes, which are the targets of over half of all commercially available drugs, and are not possible to visualize directly using other techniques. This work will utilize an alternating-current STM (AC-STM) that circumvents the conductivity requirement by measuring the tunneling current as an alternating bias is applied to the sample at frequencies from 0.5-20 GHz. The tunneling current has been shown to scale with surface polarizability or capacitance, providing spectroscopic as well as topographic information about molecules in the tunneling junction. This technique can be applied to make sub-nanometer topographic and spectroscopic measurements of biomolecules on surfaces. This proposal specifically aims to characterize two types of proteins on surfaces as representative examples of two classes of protein targets: 1) the copper redox protein P. aeruginosa azurin, as a model for the class of metalloproteins, 2) serotonin 5-HT1 receptor proteins specifically bound to their ligand on a surface, as models for GCPRs and other membrane-associated proteins, and as a demonstration of the ability to correlate structure with ligand-binding properties. Relevance: The ability to image the structures of single biomolecules such as proteins would have major implications for medical science ranging from disease diagnosis to drug discovery. This proposal describes the development of instrumentation and methodology that will enable structural information to be extracted from single molecules on surfaces. This capability will be applied to proteins embedded in lipid bilayers, which are the targets of over 50% of all modern drugs, and which are difficult to analyze by other means.
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