Photoactivation mechanism of a carotenoid-based photoreceptor

Abstract

Photoprotection is essential for efficient photosynthesis. Cyanobacteria have evolved a unique photoprotective mechanism mediated by a water-soluble carotenoid-based photoreceptor known as orange carotenoid protein (OCP). OCP undergoes large conformational changes in response to intense blue light, and the photoactivated OCP facilitates dissipation of excess energy via direct interaction with allophycocyanins at the phycobilisome core. However, the structural events leading up to the OCP photoactivation remain elusive at the molecular level. Here we present direct observations of light-induced structural changes in OCP captured by dynamic crystallography. Difference electron densities between the dark and illuminated states reveal widespread and concerted atomic motions that lead to altered protein-pigment interactions, displacement of secondary structures, and domain separation. Based on these crystallographic observations together with sitedirected mutagenesis, we propose a molecular mechanism for OCP light perception, in which the photochemical property of a conjugated carbonyl group is exploited. We hypothesize that the OCP photoactivation starts with keto-enol tautomerization of the essential 4-keto group in the carotenoid, which disrupts the strong hydrogen bonds between the bent chromophore and the protein moiety. Subsequent structural changes trapped in the crystal lattice offer a high-resolution glimpse of the initial molecular events as OCP begins to transition from the orange-absorbing state to the active red-absorbing state.photoreceptor | light perception | photoprotection | carotenoid-binding protein | dynamic crystallography

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

“…Rather, the rates of the red to orange back-conversion were inversely proportional to the phosphate concentration ( Supplementary Figure 3 ). These facts demonstrate that Trp-288 is not necessary for the photoswitching of OCP itself, but rather is important for the stabilization of the OCP OI intermediate during back-conversion, or for formation of the final compact OCP O state with its distinct carotenoid curvature as recently suggested by Bandara et al (36).…”

“…in the dimeric COCP characterized earlier (15). Such dimers may be additionally stabilized by cross-protein interactions of NTEs and CTDs (36). Formation of such violet forms requires intermediates, in which the carotenoid is positioned in the CTD.…”

“…Photoconversion is apparently accompanied by a reduction of the ring rotation angles at the C6-C7(C6’-C7’) single bonds of the carotenoid, which leads to an increase of the apparent conjugation length of the polyene chain, and to a relief of bending tension of the latter. As outlined by the recent study of Bandara and colleagues (36), this strained chromophore configuration in the dark-adapted state distinguishes OCP from other photoreceptors, in which strained chromophores are the initial or intermediate photoproducts on the way to the product state. Bending of the chromophore in OCP O most likely is due to the off-axis H-bond interaction of the 4-keto oxygen to the indole N-H group of Trp-288 in the CTD, while the carotenoid is otherwise tightly packed by a cavity constriction at the inter-domain interface and mainly hydrophobic and π-stacking interactions with Trp-110 in the NTD.…”

“…Bending of the chromophore in OCP O most likely is due to the off-axis H-bond interaction of the 4-keto oxygen to the indole N-H group of Trp-288 in the CTD, while the carotenoid is otherwise tightly packed by a cavity constriction at the inter-domain interface and mainly hydrophobic and π-stacking interactions with Trp-110 in the NTD. Dynamic X-ray crystallography has identified very early structural intermediates of photoconversion showing detachment of the αA-helix, initial rigid-body separation of the CTD and retraction of Tyr-201 and Trp-288 from the terminal β-ring, indicating that breaking of the specific chromophore-protein H-bonds is one of the initial triggers of photoconversion (36). At the other end of the time scale, in the range of hundreds of milliseconds to hundreds of seconds, spectroscopic monitoring of light-induced absorption or fluorescence changes has revealed the kinetic and thermodynamic aspects of the global phototransformations between OCP O and OCPR (11), as well as the influence of amino acid substitutions, or the action of FRP on these processes (20-22).…”

Ultrafast Structural Changes Decomposed from Serial Crystallographic Data

“…This necessity for decomposition presents both a challenge and a unique opportunity for dynamic crystallography, broadly defined as experimental and analytical approaches that tightly join crystallographic datasets and their metadata (Ren et al, 2013). We have developed a numerical process of deconvolution, which is able to identify and extract the dynamic signals from a large collection of datasets acquired at varying experimental conditions such as time points and temperatures (Bandara et al, 2017; Kim et al, 2017; Ren, 2016; Ren et al, 2012; Yang et al, 2011; Zeng et al, 2015). In essence, this strategy takes advantage of the structural heterogeneity, rather than preemptively avoids them as in the common practice of static protein crystallography.…”