Schlichting, I. | Search bioRxiv

bioRxiv2022

Structure-function-dynamics relationships in the peculiar Planktothrix PCC7805 OCP1: impact of his-tagging and carotenoid type.

The orange carotenoid protein (OCP) is a photoactive protein involved in cyanobacterial photoprotection. Here, we report on the functional, spectral and structural characteristics of the peculiar Planktothrix PCC7805 OCP (Plankto-OCP). We show that this OCP variant is characterized by higher photoactivation and recovery rates, and a stronger energy-quenching activity, compared to other OCP studied thus far. We characterize the effect of the functionalizing carotenoid and of his-tagging on these reactions, and identify the time scales on which these modifications affect photoactivation. The presence of a his-tag at the C-terminus has a large influence on photoactivation, thermal recovery and PBS-fluorescence quenching, and likewise for the nature of the carotenoid that additionally affects the yield and characteristics of excited states and the ns-s dynamics of photoactivated OCP. By solving the structures of Plankto-OCP in the ECN- and CAN-functionalized states, each in two closely-related crystal forms, we further unveil the molecular breathing motions that animate Plankto-OCP at the monomer and dimer levels. We finally discuss the structural changes that could explain the peculiar properties of Plankto-OCP. HighlightsO_LIComplete functional characterization of Synechocystis and Planktothrix OCP C_LIO_LIHitherto unknown structures of ECN- and CAN-functionalized Planktothrix OCP C_LIO_LIInsights into fs-s timescale photodynamics of ECN- and CAN-functionalized Synechocystis and Planktothrix OCP C_LI

biophysics↗

bioRxiv2021

A unifying perspective of the ultrafast photo-dynamics of Orange Carotenoid Protein from Synechocystis: peril of high-power excitation, existence of different S* states and influence of tagging

A substantial number of Orange Carotenoid Protein (OCP) studies have aimed to describe the evolution of singlet excited states leading to the formation of photo-activated form, OCPR. The most recent one suggests that three picosecond-lived excited states are formed after the sub-100 fs decay of the initial S2 state. The S* state which has the longest reported lifetime of a few to tens of picoseconds is considered to be the precursor of the first red photoproduct P1. Here, we report the ultrafast photo-dynamics of the OCP from Synechocystis PCC 6803, carried out using Visible-NIR femtosecond time-resolved absorption spectroscopy as a function of the excitation pulse power and wavelength. We found that a carotenoid radical cation can form even at relatively low excitation power, obscuring the determination of photo-activation yields for P1. Moreover, the comparison of green (540 nm) and blue (470 nm) excitations revealed the existence of an hitherto uncharacterized excited state, denoted as S[~], living a few tens of picoseconds and formed only upon 470 nm excitation. Since neither the P1 quantum yield nor the photo-activation speed over hundreds of seconds vary under green and blue continuous irradiation, this S[~] species is unlikely to be involved in the photo-activation mechanism leading to OCPR. We also addressed the effect of His-tagging at the N- or C-termini on excited state photo-physical properties. Differences in spectral signatures and lifetimes of the different excited states were observed, at variance with the usual assumption that His-tagging hardly influences protein dynamics and function. Altogether our results advocate for careful consideration of the excitation power and His-tag position when comparing the photo-activation of different OCP variants, and beg to revisit the notion that S* is the precursor of photoactivated OCPR. TOC O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=166 SRC="FIGDIR/small/474187v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@1064473org.highwire.dtl.DTLVardef@77e3c7org.highwire.dtl.DTLVardef@10b1e0eorg.highwire.dtl.DTLVardef@e24977_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗