Light- and pH-dependent structural changes in cyanobacteriochrome AnPixJg2

Abstract

Cyanobacteriochromes (CBCRs) are phytochrome-related photosensory proteins that play an essential role in regulating phototaxis, chromatic acclimation, and cell aggregation in cyanobacteria. Here, we apply solid-state NMR spectroscopy to the red/green GAF2 domain of the CBCR AnPixJ assembled in vitro with a uniformly 13C- and 15N-labeled bilin chromophore, tracking changes in electronic structure, geometry, and structural heterogeneity of the chromophore as well as intimate contacts between the chromophore and protein residues in the photocycle. Our data confirm that the bilin ring D is strongly twisted with respect to the B–C plane in both dark and photoproduct states. We also identify a greater structural heterogeneity of the bilin chromophore in the photoproduct than in the dark state. In addition, the binding pocket is more hydrated in the photoproduct. Observation of interfacial 1H contacts of the photoproduct chromophore, together with quantum mechanics/molecular mechanics (QM/MM)-based structural models for this photoproduct, clearly suggests the presence of a biprotonated (cationic) imidazolium side-chain for a conserved histidine residue (322) at a distance of ~2.7 Å, generalizing the recent theoretical findings that explicitly link the structural heterogeneity of the dark-state chromophore to the protonation of this specific residue. Moreover, we examine pH effects on this in vitro assembled holoprotein, showing a substantially altered electronic structure and protonation of the photoproduct chromophore even with a small pH drop from 7.8 to 7.2. Our studies provide further information regarding the light- and pH-induced changes of the chromophore and the rearrangements of the hydrogen-bonding and electrostatic interaction network around it. Possible correlations between structural heterogeneity of the chromophore, protonation of the histidine residue nearby, and hydration of the pocket in both photostates are discussed. Graphical abstract

Photocycle of a cyanobacteriochrome: a charge defect on ring C impairs conjugation in chromophore

“…Surprisingly, during the first two transitions, the neighboring carbons of rings B (C9) and C (C11) showed only minor Δ δ C changes. It is thus clear that a deprotonation and reprotonation cycle associated with the chromophore (protonation dynamics) in the Meta-R intermediate and subsequent transition, as proposed for both canonical phytochromes 6,13–15 and the red/green CBCR AnPixJg2 31 can be excluded for Slr1393g3, otherwise a drastic Δ δ C change of C9 would be observed. 33,34 Moreover, although it is widely accepted that the isomerization of the C15,C16-double bond is solely linked to the formation of the two primary intermediates, Lumi-R in the forward-reaction and Lumi-G in the backward-reaction, 21 the Δ δ C changes associated with the C15-methine carbon and D -ring carbonyl C19 in the Pr → Lumi-R photoreaction (−0.2 and +0.1 ppm, respectively) are much smaller than those seen in canonical phytochromes, 21 implying that ring D is twisted away from the B – C plane in both states.…”

“…In the Pr state, the dynamic nuclear polarization (DNP)-enhanced MAS NMR study of Cph1 has shown that the positive charge is mainly localized at ring B , not spreading over both inner rings as generally assumed. 46 Moreover, the 13 C chemical shifts of the pyrrolic carbons in Cph1 show great similarity to those of AnPixJg2 and the closely related NpR6012g4 in their respective Pr dark states, 18,31,44 suggesting that the electronic structure of the conjugated systems and charge distribution patterns of the Pr chromophores are comparable for all species. These observations imply the migration of partial positive charge into ring C during the Pr → Lumi-R transition.…”

“…This change is not in fact essential for phytochromes, 41,42 but contributes to the formation of a steric blockade that nalizes the twisted D-ring geometry of the Pg photoproduct in red/green CBCRs. 7,11,31,43 As a further consequence of the C-ring propionate bending, a concomitant twist in the C10-methine bridge would occur. Although changes of the tilt angle between rings B and C are predicted to profoundly impact on the peak absorption of the chromophore, in this case, the change appears to be too small (6.0°) for being the decisive factor for the 42 nm blue shi associated with photoproduct formation from Meta-R.…”

Transient 2D IR spectroscopy and multiscale simulations reveal vibrational couplings in the Cyanobacteriochrome Slr1393-g3

“…7 According to NMR and RR experiments, the PCB chromophore deprotonates either at the ring B or C pyrrole nitrogen, resulting in a tautomeric species where only one proton is shared between these two pyrrole rings. 71,72 In the light of these findings, we assign the 50 µs intermediate to such a deprotonated PCB species.…”

“…[65][66][67] Transient de-and re-protonation events are also typical for the photocycle of phytochromes 68,69 , and some studies identified such processes in the red/green CBCR AnPixJ. 70,71 Interestingly, pH-dependent experiments on the same sample indicated that the protonation state of the chromophore has a large affect on the vibrational spectra, but barely affects the visible absorption properties 71,72 , which might explain why the 250 µs process could not be resolved in the transient visible experiments. 7 According to NMR and RR experiments, the PCB chromophore deprotonates either at the ring B or C pyrrole nitrogen, resulting in a tautomeric species where only one proton is shared between these two pyrrole rings.…”