Curated Optogenetic Publication Database

Abstract: Photoactive yellow protein (PYP) is one of the typical light sensor proteins. Although its photoreaction has been extensively studied, no downstream partner protein has been identified to date. In this study, the intermolecular interaction dynamics observed between PYP from Rhodobacter capsulatus (Rc-PYP) and a possible downstream protein, PYP-binding protein (PBP), were investigated. It was found that UV light induced a long-lived product (pUV*), which interacts with PBP to form a stable hetero-hexamer (Complex-2). The reaction scheme for this interaction was revealed using transient absorption and transient grating methods. Time-resolved diffusion detection showed that a hetero-trimer (Complex-1) is formed transiently, which produced Complex-2 via a second-order reaction. Any other intermediates, including those from pBL, do not interact with PBP. The reaction scheme and kinetics are determined. Interestingly, long-lived Complex-2 dissociates upon excitation with blue light. These results demonstrate that Rc-PYP is a photochromic and new type of UV sensor to sense the relative intensities of UV-A and blue light.

Abstract: Recently, re-purposing of cyanobacterial photoreceptors as optogentic actuators enabled light-regulated protein expression in different host systems. These new bi-stable optogenetic tools enable interesting new applications, but their light-driven working mechanism remains largely elusive on a molecular level. Here, we study the optogenetic cyanobacteriochrome Am1-c0023g2 with isotope labeling and two dimensional infrared (2D-IR) spectroscopy. Isotope labeling allows us to isolate two site-specific carbonyl marker modes from the overwhelming mid-IR signal of the peptide backbone vibrations. Unlike conventional difference-FTIR spectroscopy, 2D-IR is sensitive to homogeneous and inhomogeneous broadening mechanisms of these two vibrational probes in the different photostates of the protein. We analyse the 2D-IR line shapes in the context of available structural models and find that they reflect the hydrogen-bonding environment of these two marker groups.

Abstract: The absorption and emission spectroscopic behavior of the photo-activated adenylate cyclase NgPAC2 from the amoeboflagellate Naegleria gruberi NEG-M strain was studied in the dark, during blue-light exposure and after blue-light exposure. The typical BLUF domain (BLUF = Blue Light sensor Using Flavin) flavin cofactor absorption and fluorescence photo-cycle dynamics was observed. For fresh samples a reversible concentration dependent protein oligomerization occurred showing up in free flavin binding and protein color center formation with increasing protein concentration. Thermal and temporal irreversible protein unfolding with loss of BLUF domain activity was investigated. Temperature dependent protein melting times and the apparent protein melting temperature were determined. The photodynamic behavior of the NgPAC2 is compared with the behavior of the previously investigated photo-activated cyclase NgPAC1 (nPAC) from the same N. gruberi NEG-M strain.

Abstract: The BlrP1 protein from the enteric bacterium Klebsiella pneumoniae consists of a BLUF and an EAL domain and may activate c-di-GMP phosphodiesterase by blue-light. The full-length protein, BlrP1, and its BLUF domain, BlrP1_BLUF, are characterized by optical absorption and emission spectroscopy. The cofactor FAD in its oxidized redox state (FADox) is brought from the dark-adapted receptor state to the 10-nm red-shifted putative signalling state by violet light exposure. The recovery to the receptor state occurs with a time constant of about 1 min. The quantum yield of signalling state formation is about 0.17 for BlrP1_BLUF and about 0.08 for BlrP1. The fluorescence efficiency of the FADox cofactor is small due to photo-induced reductive electron transfer. Prolonged light exposure converts FADox in the signalling state to the fully reduced hydroquinone form FADredH and causes low-efficient chromophore release with subsequent photo-degradation. The photo-cycle and photo-reduction dynamics in the receptor state and in the signalling state are discussed.