Combined Mutagenesis and Kinetics Characterization of the Bilin‐Binding GAF Domain of the Protein Slr1393 from the Cyanobacterium Synechocystis PCC6803

Abstract

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 (λmax =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 μs, 390 μs, and 1.5 ms) for the red-to-green conversion, and 1.2 μs, 340 μs, 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 green-absorbing 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.

Genomic Survey and Biochemical Analysis of Recombinant Candidate Cyanobacteriochromes Reveals Enrichment for Near UV/Violet Sensors in the Halotolerant and Alkaliphilic Cyanobacterium Microcoleus IPPAS B353

“…Furthermore, H-bonding networks mediated by the XI His and VII Arg residues are predicted to play important tuning roles (47). However, replacement of the IX Phe-525 of Slr1393g3 with Ala did not alter the spectrum of the variant (54). Taken together, these results fail to implicate a direct role of these residues in red/green CBCR chromophorylation or photointerconversion.…”

“…Taken together, these results fail to implicate a direct role of these residues in red/green CBCR chromophorylation or photointerconversion. Substitution variants W496I at position IV and R508N at position VII of Slr1393g3 displayed respective blue (25 nm)-and red (42 nm)-shifted photostate absorption maxima, suggesting involvement of these residues in photoproduct formation (47,54). However, the position IV "lid-Trp" is absent in the known red/green CBCRs NpR6012g3 and NpR5113g2 and is dispensable in NpR6012g4 (9,21).…”

A trapped double bond-photoisomerization intermediate in a bacterial photoreceptor

“…g3, indicates the number of the GAF domain of a CBCR that binds the chromophore) has the 113 capability to bind a bilin chromophore, however, this property, as well as the photochemical 114 features, are also preserved if Slr1393g3 is expressed independently as an autonomous protein 115 (Figure 1 b) (Xu et al, 2014). The absorbance maxima of both parental and photoproduct state are 116 sufficiently well separated allowing a nearly complete mutual light-driven conversion of one form 117 into the other, and, in addition, the photoproduct state of Slr1393g3 shows a remarkable thermal 118 stability: in the dark, the thermal re-conversion into the parental state proceeds to less than 10 % 119 over 24 h (20 °C) (Xu et al, 2014).…”

“…The absorbance maxima of both parental and photoproduct state are 116 sufficiently well separated allowing a nearly complete mutual light-driven conversion of one form 117 into the other, and, in addition, the photoproduct state of Slr1393g3 shows a remarkable thermal 118 stability: in the dark, the thermal re-conversion into the parental state proceeds to less than 10 % 119 over 24 h (20 °C) (Xu et al, 2014). 120…”

Tracking the secondary photodynamics of the green/red cyanobacteriochrome RcaE from Fremyella diplosiphon

“…In contrast to the secondary dynamics of other CBCRs, 25,[29][30][31]40 which exhibit significant spectral evolution, the extracted EADS exhibit only small changes of amplitudes and peak wavelengths, with the exception of the initial excitedstate 15Z P g * and terminal 15E P r spectra (EADS1 and EADS6). Based on this EADS analysis, the previous PP signals, and the interpretation of the raw data in Figure 2 …”