Bacteriophytochromes (BphPs) are red-light photoreceptors found in photosynthetic and nonphotosynthetic bacteria that have been recently engineered as infrared fluorescent tissue markers. Light-induced, global structural changes are proposed to originate within their covalently bound biliverdin chromophore and propagate through the protein. Classical BphPs undergo reversible photoconversion between spectrally distinct light absorbing states, red (Pr) and far-red (Pfr), respectively. RpBph3 (P3), from Rhodopseudomonas palustris, photoconverts between a Pr and a unique near-red (Pnr) light-absorbing state. Due to size and photosensitivity of BphPs, structures of the intact proteins have not been resolved by nuclear magnetic resonance and/or X-ray crystallography. Therefore, structural details about the light and dark-adapted structures of the intact BphPs are not well understood at the molecular level. We have utilized fluid cell atomic force microscopy (AFM) to investigate the domain structure of intact P3 in its light-adapted state (Pnr). By varying the concentration of the protein, deposition time, and the ionic strength of the buffer, the aggregation of P3 on a mica surface can be controlled and single dimers may be observed in a biologically relevant media. Domain resolution has been achieved for several orientations of the dimer on the surface. The structural dimensions of the dimer have been compared to a modeled BphP in its intact form generated using PyMOL software. AFM experiments are currently underway to analyze the dark-adapted state (Pr) of P3 in order to observe the anticipated structural changes. Ultimately, the goal is to use AFM and other surface analytical methods such as scanning tunneling microscopy and electron microscopy to gain new insight into the unique photochemistry of P3.

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 (HK) domain. Light-induced, global structural changes are proposed to originate within the covalently attached biliverdin chromophore, a linear tetrapyrrole, and propagate through the protein. Bphs undergo 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. The energy dissipated during Pr/Pfr photoconversion is proposed to directly impact the infrared fluorescence quantum yield. At this time, only structures of three different Bphs have been published, all of truncated proteins in their respective dark-adapted states. We have utilized scanning probe microscopy (SPM) to investigate the structure of intact Bphs in the light-adapted state in order to gain new insight into the mechanism of photoconversion and fluorescence. Scanning tunneling microscopy (STM) analysis of a pair of Bphs from photosynthetic bacterium R. palustris, RpBphP2 (P2) and RpBphP3 (P3) in their light-adapted states is presented in these proceedings. The concentration of the depositing protein has a key role in the molecular arrangements observed on the highly-ordered pyrolytic graphite (HOPG) surface. For example, at a high protein concentration, a hexagonal lattice of Bphs is observed by STM on a HOPG surface. Upon dilution, the photoreceptors self-organize into fiber-like structures on the surface. In these fibers, the dimer interface and the individual domains of the Bphs can be assigned and directly compared to a structural model of the intact, full-length proteins. In summary, SPM has potential to be an effective method for gaining new insight into Bph structure and dynamics.

Reflection-absorption infrared data are presented for H8Si8O12 clusters chemisorbed to Si(100)-2×1 and evaporated gold surfaces. Peak assignments and the implications of the local symmetry imposed upon the cluster by the surface are discussed.