Characterizing Architecture Of Red-Light Photoreceptors Using Scanning Tunneling Microscopy

Bacteriophytochromes (Bphs) are bacterial red-light photoreceptors that have been recently engineered for use as infrared (IR) fluorescent tissue markers. Bph from the photosynthetic bacterium Rhodopseudomonas palustris RpBpP3 (P3) naturally fluoresces in the IR range. IR fluorescent tissue markers are less prone to absorption and light scattering by hemoglobin and water than GFP-derived fluorescent proteins. Bphs have photosensory core module (PCM) consisting of 3 domains, PAS, GAF, and PHY.  The PHY is covalently linked to the histidine kinase (HK) effector domain, which through interaction with other proteins in the cell converts light signals to chemical signals. Currently, there is limited knowledge of the signal mechanisms due to the lack of structural information of intact Bphs composed of PCM and HK domain. Our goal is to characterize the truncated (lacking HK domain) and the full-length variants of P3 using scanning tunneling microscopy (STM).  We have obtained images showing intact protein dimers on a graphite surface with the predicted domain configurations for both P3 variants.  Furthermore, our images reveal a significant difference in length between truncated and full-length P3. Changing the tunneling current and bias voltage leads to expected changes in resolution of both P3 variants.  Future plans include using hydrophilic surfaces for this analysis such as gold and/or a self-assembled monolayer functionalized gold. We predict that on new surfaces the Bph is likely to have a different orientation enabling us to further characterize the P3 structure.