Imaging Bacterial Photoreceptors by Scanning Tunneling Microscopy

Scanning tunneling microscopy (STM) enables high resolution imaging of conductive surfaces at the molecular and atomic scales. Biological molecules, which in general lack the rigidity and conductivity to be studied by STM, can be adsorbed onto the surface of conductive materials, such as highly oriented pyrrolytic graphite (HOPG), and imaged in simple experimental conditions. Bacteriophytochromes (Bphs) are red-light photoreceptors found in photosynthetic and non-photosynthetic bacteria that have been engineered into infrared fluorescent protein markers. Bphs are composed of a photosensory module that is covalently linked to an effector/regulatory module, usually a histidine kinase domain. Light-induced, global structural changes are proposed to originate within the covalently attached biliverdin chromophore, a linear tetrapyrrole, and propagate throughout the protein. Bphs undergo a unique, reversible photoconversion between two distinct red and far-red light absorbing states, denoted Pr and Pfr respectively. For most Bphs, Pr is the dark-adapted state, and Pfr is the light-adapted state. Bphs’ large size and sensitivity to light lead to difficulties that currently prohibit structural characterization by NMR and X-ray crystallography. Our goal is to utilize STM to analyze Bphs in their respective light- and dark-adapted conformations in order to gain new insight into the mechanism of photoconversion and fluorescence. Bphs from photosynthetic bacterium R. palustris, RpBphP2 (P2) and RpBphP3 (P3), are imaged individually on HOPG in both truncated and full-length variants and directly compared to existing cryo-electron microscopy data for global conformational changes.