Scanning Probe Microscopy of Bacterial Red Light Photoreceptors
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Scanning Probe Microscopy of Bacterial Red Light Photoreceptors Fernando G. Tobias1, Anna Gawedzka1, 2, Max S. Goldmeier1, Alexandra C. Sakols1, Emina A. Stojković2, Stefan Tsonchev1, and Kenneth T. Nicholson1 1
Northeastern Illinois University, Department of Chemistry, 5500 N. St. Louis Ave., Chicago, IL 60625, USA 2 Northeastern Illinois University, Department of Biology, 5500 N. St. Louis Ave., Chicago, IL 60625, USA ABSTRACT Bacteriophytochromes (Bphs) are red-light photoreceptors found in photosynthetic and nonphotosynthetic 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 darkadapted 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 fiberlike 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. INTRODUCTION Photoreceptors are regulators of light-driven signal transduction pathways that play a key role in essential processes from cell division to survival. Bacteriophytochromes (Bphs) are bacterial red-light photoreceptors that are homologous to phytochrome-like proteins found in fungi and plants. Bphs are composed of a photosensory core module (PCM) that is covalently linked to an output or signaling-effector module such as a histidine kinase (HK) domain [1]. The PCM domains are PAS - Per, Arnt and Sim, [2] GAF - cGMP-phosphodiesterase, adenylyl cyclase and FhlA, [3, 4] and PHY – phytochrome specific domain [5]. While an o
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