MAS NMR on a Red/Far-Red Photochromic Cyanobacteriochrome All2699 from Nostoc

Abstract

Unlike canonical phytochromes, the GAF domain of cyanobacteriochromes (CBCRs) can bind bilins autonomously and is sufficient for functional photocycles. Despite the astonishing spectral diversity of CBCRs, the GAF1 domain of the three-GAF-domain photoreceptor all2699 from the cyanobacterium Nostoc 7120 is the only CBCR-GAF known that converts from a red-absorbing (Pr) dark state to a far-red-absorbing (Pfr) photoproduct, analogous to the more conservative phytochromes. Here we report a solid-state NMR spectroscopic study of all2699g1 in its Pr state. Conclusive NMR evidence unveils a particular stereochemical heterogeneity at the tetrahedral C31 atom, whereas the crystal structure shows exclusively the R-stereochemistry at this chiral center. Additional NMR experiments were performed on a construct comprising the GAF1 and GAF2 domains of all2699, showing a greater precision in the chromophore–protein interactions in the GAF1-2 construct. A 3D Pr structural model of the all2699g1-2 construct predicts a tongue-like region extending from the GAF2 domain (akin to canonical phytochromes) in the direction of the chromophore, shielding it from the solvent. In addition, this stabilizing element allows exclusively the R-stereochemistry for the chromophore-protein linkage. Site-directed mutagenesis performed on three conserved motifs in the hairpin-like tip confirms the interaction of the tongue region with the GAF1-bound chromophore.

Effect of the PHY Domain on the Photoisomerization Step of the Forward Pr→Pfr Conversion of a Knotless Phytochrome

“…Thus, the observed spectral shift of P fr appears to be inducedb yi nteractions of the PCB chromophore with the "tongue" region of the g2 domain in g1g2. [32,34] Interestingly, we find that the quantum yield (QY) of the P r !P fr transition is increased from~8% to~13 %( as imilarQ Yi so bserved in the related Cph2 [35] ). Hence, it follows that while the presenceo f the g2 domain does not directly affect the spectralp roperties of the P r form it does affect its photochemistry.…”

“…Recently,i tw as reported that in g1 and in g1g2 there exists ab road distribution of ground state subconformations that rapidly interconvert in solution. [32,48] These subconformations could partially contribute to the observed distributed character of the P r * decay kinetics. However,t heir rapid interconversion denotes that they are separated by low energetic barriers, and thus cannot explain the large exciteds tate barriert hat determines the relatively slow P r * decay kinetics (100 ps timescale).…”

“…The signs of these signals indicate the orientation of the peripheral rings Aa nd Dw ith respect to the co-plane of rings B and C. [8,36] This overall orientation appearsu naffected by the presence of the "tongue", whichi si nl ine with the slight changes in the dihedrala ngle of rings Aa nd Do bserved by NMR. [32] In comparison, other phytochromes like Cph1 [37] and Cph2 [35] exhibit as imilar sign change of the Q-band, while most CBCRs and bacteriophytochromes show no sign change upon switching. [36,[38][39][40] Ultrafast dynamics of P r * and formation of Lumi-R…”

“…the "tongue" region of the PHY domain with the chromophore-binding pocket in the GAF domain limits the conformational space of rings Aa nd Do ft he PCB and drives the Tyr142 residue away from the chromophorea sc ompared with g1. [32] Therefore, it appearsa si ft he "tongue" region keeps the chromophorea nd the binding pocket in am ore reactive conformation. This decreases the extent of protein conformationr eorganization required for facilitating the isomerization of the PCB chromophore and results in an acceleratedP r * decay kinetics ( Figure 5) andamore efficient P r !P fr photoconversion (less energy being lost on nonproductive degrees of freedom).…”

“…[8] Here, we report on the forward (P r !P fr )p hotoisomerization dynamics of the complete knotless phytochromeA ll2699g1g2( structurally similar to Cph2). [31,32] The homology model of All2699g1g2 [32] (Figure 1) shows that the "tongue" region of the g2 domain interacts with the chromophore bound to g1, just like the PHYd omains of canonical phytochromes. Therefore, the g1g2 construct provides au nique opportunity to directly evaluate the role of the "tongue" and thereby of the protein matrix in the photoisomerization of phytochromes.F urthermore, our resultsg ive insight into the photochemistry of knotless phytochromes, as their ultrafast dynamics has not been studied previously.…”