Blue News Update: BODIPY-GTP Binds to the Blue-Light Receptor YtvA While GTP Does Not

Abstract

Light is an important environmental factor for almost all organisms. It is mainly used as an energy source but it is also a key factor for the regulation of multiple cellular functions. Light as the extracellular stimulus is thereby converted into an intracellular signal by photoreceptors that act as signal transducers. The blue-light receptor YtvA, a bacterial counterpart of plant phototropins, is involved in the stress response of Bacillus subtilis. The mechanism behind its activation, however, remains unknown. It was suggested based on fluorescence spectroscopic studies that YtvA function involves GTP binding and that this interaction is altered by absorption of light. We have investigated this interaction by several biophysical methods and show here using fluorescence spectroscopy, ITC titrations, and three NMR spectroscopic assays that while YtvA interacts with BODIPY-GTP as a fluorescent GTP analogue originally used for the detection of GTP binding, it does not bind GTP.

LOV Takes a Pick: Thermodynamic and Structural Aspects of the Flavin-LOV-Interaction of the Blue-Light Sensitive Photoreceptor YtvA from Bacillus subtilis

“…Expression of 15 N-labeled YLOV was done similar to the method previously described for the expression of the 15 N-labeled YtvA-STAS domain, except the expression temperature, which was kept at 25 °C. For details see Dorn et al [ 50 ]. Recombinant expression of unlabeled YLOV was also carried out in a high cell density fermentation system (FedBatch Pro, DASGIP, Germany) using T7-Express Rosetta2 cells as expression host (New England Biolabs GmbH, Frankfurt a.M., Germany).…”

“…TEV cleavage and further purification of YLOV was performed as described earlier [ 50 ] except that buffer B was used in the size exclusion chromatography. Riboflavin incorporated in heterologously expressed YtvA and YLOV not used for apoprotein production was exchanged against FMN.…”

“…The half-life of the photoactive state was calculated by averaging the corresponding values obtained from the single exponential fits of both relaxation curves. For photo-conversion and maintenance of the photostationary state, the previously described fiber-coupled light source was used [ 50 ]. Calculation of chemical shift differences (CSD) and crosspeak intensity ratios from 2D heteronuclear NMR spectra was done using CCPNMR analysis v.2.2 software [ 59 ].…”

“…The origin of the other signal (B) is unknown. To obtain the lit-state spectrum, sample was illuminated using our previously described fiber coupled light source [ 50 ]. Spectra were acquired at 27°C on a 600 MHz NMR spectrometer (Bruker Avance III) equipped with a 5 mm triple resonance PFG (z-axis) cryo probe head.…”

A structural model for the full-length blue light-sensing protein YtvA from Bacillus subtilis, based on EPR spectroscopy

“…[4][5][6] YtvA is a relatively small protein of only 261 amino acids and is composed of two domains, the light-sensing LOV domain and a putative signalling STAS domain (STAS, Sulfate Transporter and Anti-Sigma antagonist), both domains being connected by a short helical linker. Despite intensive research into the structure and the function of YtvA, [7][8][9][10][11] structural information on the full-length protein is still limited. 6,8,12 Detailed structural knowledge is clearly required, as information on a monomeric or dimeric state or on the domain arrangement of these receptors is essential for an understanding of their biological function.…”