Characterization of a Unique Bacterial Photoreceptor in the Light-Adapted State using Atomic Force Microscopy

Our research utilizes an interdisciplinary approach which looks at unique family of signaling proteins known as bacteriophytochromes (Bphs), bacterial red-light photoreceptors. Bphs contain a photosensory module covalently linked to a signal effector domain, histidine kinase (HK).  Upon absorption of a photon in the appropriate wavelength range, Bphs are proposed to undergo photoconversion with concomitant global structural changes. To date, limited structural information exists on Bphs due to protein size and current inability to obtain crystals of intact proteins in the respective dark and light-adapted states. Using atomic force microscopy (AFM) we have studied the structure of RpBphP3 (P3) from the photosynthetic bacterium Rhodopseudomonas palustris on mica. Most Bphs undergo reversible photoconversion between the red and far-red light-absorbing forms Pr and Pfr respectively; however, P3 undergoes photoconversion in the red and unique near-red light absorbing states Pr and Pnr respectively. Using a variety of approaches, such as manipulating the concentration of the protein and the ionic strength of the buffer, we have been able to image P3 through AFM in its light-adapted Pnr state. We have observed biologically relevant dimers as well as protein aggregates of various sizes on a mica surface in its light-adapted state. One future goal is to characterize P3 in Pnr state on a biocompatible surface, such as a functionalized poly-ethylene glycol (PEG), to reduce the number of aggregates.  Finally, we plan to compare the light and dark-adapted structures P3 and related Bphs.