Perspectives for in situ Scanning Tunnel Microscopic Imaging of Proteins at HOPG surfaces

We have investigated the behaviour of the four-copper fungal metalloenzyme laccase (MW~68kDa) at highly oriented pyrolytic graphite (HOPG) surfaces by ex situ and in situ STM. The four copper atoms ar suited to stimulate long-range inelastic tunnel modes through the protein. The proteins forms crystalline or amorphous structures of micro-meter lateral extension during evaporation of aqueous laccase solution at low ionic strength. Individual molecular-size structures distinct from the HOPG background, and possibly arising from tip dislodging can also be imaged. The HOPG surface cracks at certain potentials on in situ potentiostatic control and releases nm size HOPG scrap bits. These are clearly different in shape from the ex situ imaged molecular-size structures. Laccase could not, however, be imaged by in situ STM, most likely due to structural incompatibility between hydrophobic HOPG surface and the strongly negatively charged protein, and to high protein surface mobility.