Search

Proceedings of the National Academy of Sciences of the United States of America 117(5), 2432 - 2440 (2020). doi:10.1073/pnas.1910208117, Phytochromes and related photoreceptors distinguish themselves for their long-wavelength absorption and large spectral shift between parental state and photoproduct. Both features are not well understood, partly due to lack of high-resolution structural data and insufficient support from quantum-chemical calculations. The red–green switching cyanobacteriochrome Slr1393g3 shows an absorption shift larger than 110 nm. Both parental state and photoproduct could be crystallized with high resolution, together with a “hybrid” form carrying the chromophore in parental state geometry, whereas the protein remained in the photoproduct conformation. The crystal structures reveal how chromophore and protein mutually regulate their conformational changes, yielding the observed spectral shift. Quantum-chemical calculations, based on these structures, provide a deeper understanding of the spectral tuning mechanisms., Published by National Acad. of Sciences, Washington, DC

The second GAF domain of AnPixJ, AnPixJg2, a bilin-binding protein from the cyanobacterium Anabaena PCC 7120, undergoes a photoinduced interconversion between a red-absorbing state, Pr, and a green-absorbing state, Pg. Combining ultraviolet-vis (UV-vis), infrared, resonance Raman (RR), and magic-angle spinning (MAS) nuclear magnetic resonance (NMR) spectroscopy, we have studied this cyanobacteriochrome (CBCR) assembled with phycocyanobilin (PCB) either in vivo or in vitro. In both assembly routes, the spectroscopic data of the Pr state reveal nearly identical chromophore structures with a protonated (cationic) bilin. However, unlike the native (in vivo assembly) Pg photoproduct, in which the bilin retains protonation, the Pg generated from the in vitro-assembled AnPixJg2 harbors a deprotonated (neutral) bilin chromophore at pH 7.8. IR difference spectroscopy further reveals the transfer of a proton from the bilin to a side-chain carboxylate on an amino acid, probably Asp291. Besides the change in protonation state, the bilin structure is very similar in the in vitro- and in vivo-assembled Pg photoproducts. The chromophore of the in vitro Pg becomes protonated when the pH is increased to 10, presumably because of a partial reversal of protein misfolding. Most remarkably, the electronic transitions remain unchanged and are very similar to those of the native Pg. Thus, bilin protonation is not a key parameter for controlling the energies of the electronic transitions in AnPixJg2. Possible alternative molecular mechanisms for color tuning are discussed.

The protein encoded by the gene slr1393 from the cyanobacterium Synechocystis sp. PCC6803 (Slr1393) is composed of three GAF domains, a PAS domain, and a histidine kinase motif. The third GAF domain (referred to as GAF3) was previously characterized as the sole domain in this protein, being able to carry phycocyanobilin (PCB) as the chromophore and to accomplish photochemistry. GAF3 shows photochromicity, and is able to switch between a red-absorbing parental state (GAF3R, ?max = 649 nm) and a green-absorbing photoproduct state (GAF3G, ?max = 536 nm) upon appropriate irradiation. In this study we have determined the photochemical quantum yields for the interconversion between both forms using two methods: an "absolute" method and a reference-based control. The latter is a comparative procedure which exploits a well-characterized blue-light photoreceptor, YtvA from Bacillus subtilis, and the cyanobacterial phytochrome Cph1 as actinometers. The former is an ad hoc developed, four laser-based setup where two cw lasers provide the pump beams to induce photoswitching (red to green and green to red, respectively) and two cw lasers simultaneously monitor the appearance and disappearance of the two species. Interestingly, fit analysis of the recorded transient absorbance changes provided a quantum yield for the green -> red conversion (?0.3) at least three times larger than for the red -> green conversion (?0.08). These data are in agreement with the results from the comparative method documenting the usefulness of the 'direct' method developed here for quantum yields' determination. The light-induced switching capability of this photochromic protein allowed measuring the kinetics of GAF3 immobilized on a glass plate, and within living, overexpressing Escherichia coli cells.

The gene slr1393 from Synechocystis sp. PCC6803 encodes a protein composed of three GAF domains, a PAS domain, and a histidine kinase domain. GAF3 is the sole domain able to bind phycocyanobilin (PCB) as chromophore and to accomplish photochemistry: switching between a red-absorbing parental and a green-absorbing photoproduct state (lmax=649 and 536 nm, respectively). Conversions in both directions were followed by time-resolved absorption spectroscopy with the separately expressed GAF3 domain of Slr1393. Global fit analysis of the recorded absorbance changes yielded three lifetimes (3.2 ms, 390 ms, and 1.5 ms) for the red-to-green conversion, and 1.2 ms, 340 ms, and 1 ms for the green-to-red conversion. In addition to the wild-type (WT) protein, 24 mutated proteins were studied spectroscopically. The design of these site-directed mutations was based on sequence alignments with related proteins and by employing the crystal structure of AnPixJg2 (PDB ID: 3W2Z), a Slr1393 orthologous from Anabaena sp.PCC7120. The structure of AnPixJg2 was also used as template for model building, thus confirming the strong structural similarity between the proteins, and for identifying amino acids to target for mutagenesis. Only amino acids in close proximity to the chromophore were exchanged, as these were considered likely to have an impact on the spectral and dynamic properties. Three groups of mutants were found: some showed absorption features similar to the WT protein, a second group showed modified absorbance properties, and the third group had lost the ability to bind the chromophore. The most unexpected result was obtained for the exchange at residue 532 (N532Y). In vivo assembly yielded a red-absorbing, WT-like protein. Irradiation, however, not only converted it into the greenabsorbing form, but also produced a 660 nm, further-red-shifted absorbance band. This photoproduct was fully reversible to the parental form upon green light irradiation. Fil: Xu, Xiu Ling. Max-Planck-Institute for Chemical Energy Conversion; Alemania Fil: Gutt, Alexander. Max-Planck-Institute for Chemical Energy Conversion; Alemania Fil: Mechelke, Jonas. Max-Planck-Institute for Chemical Energy Conversion; Alemania Fil: Raffelberg, Sarah. Max-Planck-Institute for Chemical Energy Conversion; Alemania Fil: Tang, Kun. Max-Planck-Institute for Chemical Energy Conversion; Alemania. Huazhong Agricultural University; República de China Fil: Miao, Dan. Huazhong Agricultural University; República de China Fil: Valle, Lorena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Investigaciones y Transferencia de Santiago del Estero. Universidad Nacional de Santiago del Estero. Centro de Investigaciones y Transferencia de Santiago del Estero; Argentina. Universidad Nacional de Santiago del Estero. Facultad de Agronomía y Agroindustrias; Argentina Fil: Borsarelli, Claudio Darío. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Investigaciones y Transferencia de Santiago del Estero. Universidad Nacional de Santiago del Estero. Centro de Investigaciones y Transferencia de Santiago del Estero; Argentina. Universidad Nacional de Santiago del Estero. Facultad de Agronomía y Agroindustrias; Argentina Fil: Zhao, Kai Hong. Huazhong Agricultural University; República de China Fil: Gärtner, Wolfgang. Max-Planck-Institute for Chemical Energy Conversion; Alemania