Structural Characterization Of Red Light Photoreceptors Using Atomic Force Microscopy

Bacteriophytochromes (Bphs) are red-light photoreceptors found in photosynthetic and non-photosynthetic bacteria with homologs in fungi and plants that have recently been engineered as infrared fluorescent tissue markers. All Bphs naturally exist as dimers. Light-induced, global structural changes originate within the covalently attached chromophore biliverdin, and propagate through the rest of the protein. These changes are believed to be reversible during the protein’s photoconversion between two spectrally distinct light-absorbing states. RpBphP3 (P3), from Rhodopseudomonas palustris, undergoes reversible photoconversion between red (Pr) and a unique near-red (Pnr) light-absorbing states and is naturally fluorescent. Due to the size and light sensitivity of Bphs, structures of intact proteins in their light-adapted states have not been solved by NMR and/or X-ray crystallography. Therefore, we have utilized fluid cell atomic force microscopy (AFM) to investigate the structure of P3 in its light-adapted state on mica. By using buffers of varied ionic strength, we have been able to minimize the aggregation of P3 on the surface and observe single dimers in a biologically relevant media. Currently, we are using Gwyddion, a program that can create an averaged AFM image of a single molecule, in order to improve the domain resolution and complete a dimensional analysis of P3. Future work involves imaging intact P3 in its dark-adapted state. Our goal is to gain insight into the mechanism of the unique P3 photoconversion and fluorescence, which may be used to design a novel, biologically inert, infrared fluorescent tissue marker.