A Molecular Mechanism for Nonphotochemical Quenching in Cyanobacteria

Abstract

The cyanobacterial Orange Carotenoid Protein (OCP) protects photosynthetic cyanobacteria from photodamage by dissipating excess excitation energy collected by phycobilisomes (PBS) as heat. Dissociation of the PBS-OCP complex in vivo is facilitated by another protein known as the Fluorescence Recovery Protein (FRP), which primarily exists as a dimeric complex. We used various mass spectrometry(MS)-based techniques to investigate the molecular mechanism of this FRP-mediated process. FRP in the dimeric state (dFRP) retains its high affinity to the C-terminal domain (CTD) of OCP in the red state (OCPr). Site-directed mutagenesis and native MS suggest the head region on FRP is a binding candidate to OCP. After attachment to the CTD, the conformational changes of dFRP enable dFRP to bridge the two domains, facilitating the reversion of OCPr into the orange state (OCPo) accompanied by a structural rearrangement of dFRP. Interestingly, we found a mutual response between FRP and OCP; that is, FRP and OCPr destabilize each other, whereas FRP and OCPo stabilize each other. A detailed mechanism of FRP function is proposed on the basis of the experimental results.

The photocycle of orange carotenoid protein conceals distinct intermediates and asynchronous changes in the carotenoid and protein components

“…These facts might indicate that in the intermediate OCP RI , the αA helix of the NTE could already detach from the CTD uncovering the FRP binding site (22). A possible explanation of this phenomenon could be slow detachment of FRP from OCP, which occurs slower than the changes of carotenoid absorption on the timescale exceeding intervals between the excitation laser pulses in our experiment – 1 s, or due to FRP binding to OCP in the orange state, which we observed at high concentrations (21, 32). Although we support the idea of a secondary FRP binding site in the NTD of OCP (32), it seems reasonable to assume that the mechanism of FRP action is not limited to trivial coupling of the OCP domains.…”

“…A possible explanation of this phenomenon could be slow detachment of FRP from OCP, which occurs slower than the changes of carotenoid absorption on the timescale exceeding intervals between the excitation laser pulses in our experiment – 1 s, or due to FRP binding to OCP in the orange state, which we observed at high concentrations (21, 32). Although we support the idea of a secondary FRP binding site in the NTD of OCP (32), it seems reasonable to assume that the mechanism of FRP action is not limited to trivial coupling of the OCP domains. If this would be the case, we would have expected to see a reduction of the yield of the slow component for ΔNTE OCP in the presence of FRP, as observed in experiments with phosphate; however, our data indicate that strong binding of FRP to ΔNTE OCP does not prohibit formation of the OCP R state with separated domains ( Figure 1 ).…”

“…The photoprotective mechanism in Synechocystis is terminated by the action of the Fluorescence Recovery Protein (FRP), which promotes dissociation of OCP R from the PBs and accelerates recovery to the OCP O state (29-31). Binding of FRP occurs to the CTD, most likely at a site covered in OCP O by the αA-helix, although some secondary, as yet unidentified, site might necessarily exist on the NTD or the interdomain linker as well to enable reversion of domain separation and back-sliding of the carotenoid cofactor into its initial position (22, 32). Spectral and structural changes of the protein and the carotenoid upon photoexcitation have been studied by multiple spectroscopic approaches (14, 28, 33, 34); however, main spectral and structural intermediates within the OCP photocycle (if existent) are still elusive, as is the case for the atomic structure of the most important, biologically active OCP R state.…”

Deletion of the short N‐terminal extension in OCP reveals the main site for FRP binding

“…Blankenship and co-workers very recently reported interesting details of the molecular mechanism of interaction of FRP and OCP obtained by chemical crosslinking and native mass-spectrometry; the study supports FRP monomerization and FRP-mediated bridging the two OCP domains during the interaction with OCP [32].…”

Site, trigger, quenching mechanism and recovery of non-photochemical quenching in cyanobacteria: recent updates

“…The Blankenship group (Lu et al 2016;Lu et al 2017) suggests that dimeric-FRP first binds with the CTD of OCP r and induces conformational changes in dimeric-FRP that enabls its binding to the NTD domain. This, in turn, induces monomerization of dimeric-FRP, bridging and pulling of the CTD and NTD together and thereby promoting the conversionl of OCP r in to OCP o .…”