Molecular mechanisms of light harvesting in the minor antenna CP29 in near-native membrane lipidic environment

Abstract

CP29, a chlorophyll (Chl) a/ b-xanthophyll binding protein, bridges energy transfer between the major LHCII antenna complexes and Photosystem II reaction centres. It hosts one of the two identified quenching sites making it crucial for regulated photoprotection mechanisms. CP29 photophysics has been so far studied on the purified protein in detergent solutions, since spectrally overlapping signals affect in vivo measurements. However, the protein in detergent assumes non-native conformations compared to its physiological state in the thylakoid membrane. Here, we report a detailed photophysical study on CP29 inserted in discoidal lipid bilayers, known as nanodiscs, which mimic the native membrane environment. Using picosecond time-resolved fluorescence and femtosecond transient absorption (TA), we observed shortening of the Chl fluorescence lifetime with a decrease of the carotenoid triplet formation yield for CP29 in nanodiscs as compared to the protein in detergent. Global analysis of TA data suggests a 1Chl* quenching mechanism dependent on excitation energy transfer to a carotenoid dark state, likely the proposed S*, which is believed to be formed due to a carotenoid conformational change affecting the S1 state. We suggest that the accessibility of the S* state in different local environments plays a key role in determining the quenching of Chl excited states. In vivo, non-photochemical quenching is activated by de-epoxidation of violaxanthin into zeaxanthin. CP29-Zeaxanthin in nanodiscs further shortens Chl lifetime, which underlines the critical role of zeaxanthin in modulating photoprotection activity.

Site-Directed Mutagenesis of the Chlorophyll-Binding Sites Modulates Excited-State Lifetime and Chlorophyll–Xanthophyll Energy Transfer in the Monomeric Light-Harvesting Complex CP29

“…Yet, the difference spectra, shown in Figure S3, display derivative shapes, both in the Soret and Q y regions, suggesting that the lipid environment produced changes in pigment–pigment interactions, similar for all the nanodisc assemblies (Supplementary Figure S4), which result in positive peaks at 460 and 663 nm and negative peaks at 480 and 678 nm. This observation is consistent with our previous comparison of CP29 WT in detergent and nanodiscs …”

“…The DAS for CP29 WT are shown in Figure a,b, and the energy level diagrams illustrating the main photophysical processes at different time delays are shown in Figure S13. The processes have been described in detail in our previous report . Briefly, the 614 fs DAS exhibits a negative signal at 470 nm (B band) and 640 nm (Q y band) corresponding to the decay of Chl b GSB, and a negative signal at 494 nm due to the Car GSB decay, while the positive signal at 680 nm corresponds to an increase in the GSB of the Q y band of Chl a due to the EET from Chl b .…”

The origin of pigment-binding differences in CP29 and LHCII: the role of protein structure and dynamics

“…These data therefore show the distortions that detergent micelles can induce in LHCs and prompt the use of more native environments when studying them, such as e.g. liposomes [77] or nanodics [78][79][80].…”

Role of Efficient Charge Transfer at the Interface between Mixed-Phase Copper-Cuprous Oxide and Conducting Polymer Nanostructures for Photocatalytic Water Splitting

“…The absorption properties of the nanostructures were determined from UV–visible diffuse reflectance spectra (DRS) by using a UV-3600 Shimadzu spectrophotometer. A home-built setup was employed to study the femtosecond transient absorption (TA) measurements, which was based on a regeneratively amplified Ti:sapphire laser (Libra, Coherent, 100 fs pulses at 800 nm and 2 kHz repetition rate). , In order to obtain a 400 nm pump, second-harmonic generation was performed using a barium borate crystal. A broad-band probe beam (450–750 nm) was obtained using white-light continuum generation by focusing 800 nm pulses on a 2 mm thick sapphire crystal, and appropriate filters were used to remove the residuals of fundamentals.…”