The cyanobacterial Fluorescence Recovery Protein has two distinct activities: Orange Carotenoid Protein amino acids involved in FRP interaction

    The cyanobacterial Fluorescence Recovery Protein has two distinct activities: Orange Carotenoid Protein amino acids involved in FRP interaction Adrien Thurotte, C´eline Bourcier de Carbon, Adj´el´e Wilson, L´ea Talbot, Sandrine Cot, Rocio L´opez-Igual, Diana Kirilovsky PII: DOI: Reference:

S0005-2728(17)30025-7 doi:10.1016/j.bbabio.2017.02.003 BBABIO 47781

To appear in:

BBA - Bioenergetics

Received date: Revised date: Accepted date:

14 December 2016 17 January 2017 5 February 2017

Please cite this article as: Adrien Thurotte, C´eline Bourcier de Carbon, Adj´el´e Wilson, L´ea Talbot, Sandrine Cot, Rocio L´ opez-Igual, Diana Kirilovsky, The cyanobacterial Fluorescence Recovery Protein has two distinct activities: Orange Carotenoid Protein amino acids involved in FRP interaction, BBA - Bioenergetics (2017), doi:10.1016/j.bbabio.2017.02.003

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The cyanobacterial Fluorescence Recovery Protein has two distinct activities: Orange

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Carotenoid Protein amino acids involved in FRP interaction

Adrien Thurotte1,2, Céline Bourcier de Carbon2,3, Adjélé Wilson1,2, Léa Talbot1,2,

Institute for Integrative Biology of the Cell (I2BC), CNRS, CEA, Université Paris-Sud,

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Sandrine Cot1,2, Rocio López-Igual1,2, Diana Kirilovsky1,2

Université Paris-Saclay, 91198 Gif sur Yvette, France Institut de Biologie et Technologies de Saclay (iBiTec-S), Commissariat à l’Energie

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Phycosource, 13 boulevard de l'Hautil, 95092 Cergy Cedex, France

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Atomique (CEA), 91191 Gif-sur-Yvette, France.

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Corresponding author: Diana Kirilovsky, [email protected], Tel: 33 1 69089571; Fax: 33 1 69088717 The current address of AT : Institut für Pharmazie und Biochemie, Johannes Gutenberg-Universität Mainz, 55128 Mainz, Germany. The current address of RLI: Institut Pasteur, Unité Plasticité du Génome Bactérien, Département Génomes et Génétique, CNRS, Unité Mixte de Recherche 3525, F-75015 Paris, France

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Abstract

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To deal with fluctuating light condition, cyanobacteria have developed a photoprotective mechanism which, under high light conditions, decreases the energy arriving at the photochemical centers. It relies on a photoswitch, the Orange

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Carotenoid Protein (OCP). Once photoactivated, OCP binds to the light harvesting

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antenna, the phycobilisome (PBS), and triggers the thermal dissipation of the excess energy absorbed. Deactivation of the photoprotective mechanism requires the

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intervention of a third partner, the Fluorescence Recovery Protein (FRP). FRP by

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interacting with the photoactivated OCP accelerates its conversion to the non-active

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form and its detachment from the phycobilisome. We have studied the interaction of FRP with free and phycobilisome-bound OCP. Several OCP variants were constructed

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and characterized. In this article we show that OCP amino acid F299 is essential and D220 important for OCP deactivation mediated by FRP. Mutations of these amino acids did not affect FRP activity as helper to detach OCP from phycobilisomes. In addition, while mutated R60L FRP is inactive on OCP deactivation, its activity on the detachment of the OCP from the phycobilisomes is not affected. Thus, our results demonstrate that FRP has two distinct activities: it accelerates OCP detachment from phycobilisomes and then it helps deactivation of the OCP. They also suggest that different OCP and FRP amino acids could be involved in these two activities. 2

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Key words: Cyanobacteria, Fluorescence Recovery Protein, Non-photochemical

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quenching, Orange Carotenoid Protein, photoprotection, phycobilisome

High Lights:

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FRP has two distinct activities: splits OCP-PBS complex and deactivates OCP FRP mediated OCP deactivation requires D220 and F299 OCP residues

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Mutation of these amino acids does not hinder FRP interaction with bound OCP

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The distinct activities of FRP could involve different amino acids in both OCP and FRP

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1. INTRODUCTION

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Light is essential for photosynthetic organisms that convert solar energy into chemical energy and reducing power needed for cellular metabolism. However, too much light could be lethal. Photosynthesis cannot control the incoming flux of its

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substrate: light. The absorption of a photon by the light harvesting complexes

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(antennae) cannot be switched off and when the photon flux exceeds the photoconverting capacity of the cell, the entire photosynthetic chain gets congested. This

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promotes the accumulation of harmful high energy reactive intermediates including

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singlet oxygen and oxygen radicals (ROS). To avoid this, both the quantity of energy

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arriving at the reaction centers and the photosynthetic production of ATP and NADPH are continuously adjusted to match the rate of their consumption by the Calvin-

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Benson cycle and by downstream reactions for carbon and nitrogen assimilation. These adjustments are possible because photosynthetic cells possess delicate mechanisms that sense the quality and quantity of incident light and tune the pigment and/or protein composition of photosynthetic light-harvesting complexes to adjust light absorption and utilization. The organization of the photosystems and light harvesting complexes is modified to limit over-excitation by different mechanisms. One of them increases thermal dissipation of absorbed energy at the level of antennae to decrease the energy arriving at reaction centers. In cyanobacteria, this 4

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mechanism is regulated by light intensity via the photoactive and soluble light sensor

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carotenoid protein, the Orange Carotenoid Protein (OCP) (reviews [1-3]). Once

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activated by strong light, OCP interacts with the phycobilisome (PBS), the

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cyanobacterial extramembrane antenna, and induces thermal dissipation of excess energy absorbed by them [4-6]. This decreases the energy arriving at the reaction

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centers and quenches PBS fluorescence [5, 7-9].

The OCP has two different globular domains: an α-helical N-terminal domain

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(NTD) and a mixed α-helical/β-sheet C-terminal domain (CTD) joined by a flexible

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linker loop [10, 11] (Figure 1). The keto-carotenoid 3’-hydroxyechinenone (hECN)

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spans both domains. Its keto group is H-bonded to Y201 and W288. In the inactive dark orange OCP (OCPo), strong interactions occurring on the one hand in the central

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interface between the two domains and on the other hand between the N-terminal arm and the CTD stabilize a tight globular form [10, 11]. Absorption of strong bluegreen light (or white light) by hECN induces the conversion of the OCPo into the active red OCP (OCPr) [6]. The photoactivation involves conformational changes of both carotenoid and protein [6, 12-15]. Upon absorption of light by the carotenoid, the Hbonds with Y201 and W288 are broken and the carotenoid is free to translocate to its position in OCPr [13, 16, 17]. The changes in CTD specific amino acids as well as in conserved water molecules propagate the signal to the surface of the protein leading 5

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to the detachment of the N-terminal arm from the CTD and the unfolding of the

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buried A- helix [12, 14, 15, 18]. Consequently, OCP opens and interdomain

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interactions (including the salt bridge R155-E244) are broken, water molecules

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penetrate in the interfaces between the NTD and CTD domains and the carotenoid

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undergoes a large translocation (12 Å) [12-15, 18, 19].

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In the OCPr, the CTD and NTD are spatially separated and the carotenoid is completely buried in the NTD [12, 13]. The secondary structures of the NTD and CTD

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domains remain largely unchanged with the exception of the A- helix in OCPr [12,

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13]. The carotenoid binding pocket in CTD remains intact and empty, ready to

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receive the carotenoid back when the protein is deactivated (red to orange form conversion) [12]. The cavity is probably protected from exterior attack by a loop

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joining 1 and M in the CTD [12].

OCPr is the only form able to interact with phycobilisomes [4]. The PBS, which is composed by phycobiliproteins and linker proteins, is organized as a core from which rods radiate (reviews [20-22] ). In the model cyanobacterium Synechocystis PCC 6803 (thereafter Synechocystis), the rods are formed by 3 hexamers of phycocyanin (PC) and the core is formed by 2 basal cylinders and 1 upper cylinder 6

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containing 4 trimers of allophycocyanins (APCs). The linker protein CpcG stabilizes the

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binding of the rods to the core. The upper core cylinder is formed by trimers of APC-

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APC heterodimers with maximal fluorescence emission at 660 nm (APC660, blue). In

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one of the external trimers of the basal cylinders, the APC subunit is replaced by a special APC-like subunit called ApcD. In the neighboring trimer one  subunit is

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replaced by ApcF, a APC-like subunit, and one  subunit is replaced by the Nterminal domain of ApcE, an APC-like domain. The bilins attached to ApcD and ApcE

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which have a maximal fluorescence at 680 nm, are the PBS terminal energy acceptors

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and they transfer the absorbed energy to the photosystems (APC680). In each

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cylinder the 2 external trimers are stabilized by the ApcC linker protein. ApcE is also essential for the stabilization of the PBS core and for binding of PBS to the

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membrane. When the OCPr is attached to the PBS, the NTD is buried between two APC trimers of one basal cylinder [23, 24]. Recent results strongly suggest that the

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NTD is situated between the ApcD containing trimer and the ApcF-ApcE containing trimer [23]. At least in vitro, the isolated NTD (without the CTD) is able to bind PBS and to induce fluorescence and excitation energy quenching [13, 25].

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Once light intensity decreases, the soluble Fluorescence Recovery Protein

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(FRP) is needed to recover from the energy-dissipative state [26]. Almost all the OCP

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containing cyanobacteria strains also contain an frp gene [26-28]. The FRP is a soluble

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-helical protein of 13kDa that does not bind any chromophore. The oligomeric state of the protein is still under debate. In crystal form, the FRP was found as a dimer and

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as a tetramer [28]. In solution, it appears mostly as a dimer but tetramers are also detected [28, 29]. The tetramer could be an inactive form or an isolation artifact.

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Most certainly, recombinant FRP is unstable and has a tendency to form a tetramer prior to aggregation and precipitation [26, 28, 30]. FRP accelerates the conversion of

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OCPr to OCPo by interacting with the CTD of the OCPr [26, 28]. In addition it seems to help the detachment of OCPr from PBS [4]. FRP attaches to OCP in its dimeric form.

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Recent results strongly suggest that once attached (or during attachment) to OCP, FRP monomerizes [29]. In the dimer, on one side, there is a patch of amino acids that

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are 100% identical in all FRPs [28]. Mutations of some of these amino acids largely affect FRP activity. The residue R60 was demonstrated to be essential for accelerating the transition of OCPr to OCPo [28].

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Based on docking simulations, Sutter and co-workers [28], presented a model

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of FRP-OCP interaction in which the FRP dimer binds to one side of the OCP CTD [28].

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Several OCP amino acids of the CTD could be involved in this interaction, including

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D220, R229, N236, D262 and F299 [28]. Furthermore, Liu et al [14] showed that E261 and D262 undergo increased solvent accessibility upon photoactivation, when the N-

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terminal arm moves [14]. These residues form a loop connecting β2 and β3 strands in the CTD β-sheet (Figure 1). The role of all these OCP amino acids in FRP activity

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remains unclear and needs to be elucidated. In addition, it is not known how FRP accelerates the recovery of PBS fluorescence. Several possibilities are conceivable: 1)

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FRP decreases the concentration of free OCPr, 2) accelerates the conversion to OCPo of bound OCPr leading to detachment of the OCP from PBS, 3) binds to the attached

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OCPr and helps its detachment and only then accelerates its conversion to OCP o, 4) a combination of all these possibilities. Thus, the open question is whether FRP has

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only one activity (acceleration of OCPr to OCPo conversion) or two distinct activities (acceleration of OCPr to OCPo conversion and helper in detachment of OCPr from PBS). In order to further understand the FRP mechanism, we constructed and characterized 10 mutated Synechocystis OCPs and we studied the effect of WT and mutated FRPs on free and PBS-bound WT and mutated OCPs. Our results clearly demonstrate that FRP has two distinct activities and that OCP amino acids F299 and

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D220 are important for the FRP helper activity in closing the protein when it is

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converted from the red to the orange form.

2. MATERIALS and METHODS

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2.1 Plasmid construction

Construction of plasmid pCDF-OCPsynNtag carrying the slr1963 sequence with a His-

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tag sequence in the N-terminus, was described in Bourcier de Carbon et al. [31]. The

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point mutations were introduced by directed mutagenesis, using the pCDF-OCPSyn-

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Ntag plasmid as a template and mismatching primers (Eurofins) (Supplementary Fig. S1). The plasmids were used to transform E. coli cells producing echinenone (ECN) as

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described in Bourcier de Carbon et al [31]. The Crt operon containing crtB, crtE, crtI and crtY genes from Erwinia uredovora, including the crtE promoter and operon endogenous terminator, was amplified by PCR using pACCAR16CrtX plasmid [32](Gift of Prof Gerhard Sandmann) as a template and synthetic oligonucleotides Crt-pBAD (F and R) as primers (Supplementary Fig 1). The resulting PCR product was introduced into pBAD-CrtO [31]. The pBAD-CrtO plasmid containing the crtO gene under the control of the araBAD [31] promoter was digested with PmeI and XbaI to

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clone the crt operon. The PCR product was ligated to the digested pBAD-CrtO plasmid

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2.2 Gene overexpression and Protein isolation

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to create the pBAD-CrtO-Crt plasmid.

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E. coli BL21-Gold (DE3) cells from Agilent Technologies were used for OCP production. E. coli cells were transformed simultaneously with the pBAD-CrtO-Crt and pCDF-OCP

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plasmids to produce holo-OCPs. The transformed E. coli cells were grown in the

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presence of two antibiotics (ampicillin [50 mg.mL-1], and streptomycin [25 mg.mL-1])

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to maintain the different plasmids in the E. coli cells. For induction of different promoters, transformed E. coli cells were grown in Terrific Broth medium at 37°C for

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3 to 4 h until OD600 = 0.8. Then, 0.02% (w/v) arabinose was added and the culture was grown overnight at 37°C. In the morning, the cells were diluted with fresh medium and 0.02% arabinose (Sigma-Aldrich), and they were grown at 37°C until OD600 = 1 to 1.2. IPTG (Sigma-Aldrich) (0.2 mM) was then added, and cells were incubated overnight at 28°C. In the morning, the cultures were harvested and pellets were stored at -80°C until they were used [31]. E. coli BL21-Gold (DE3) cells were transformed with only pCDF-OCP to produce apo-OCP. The cells were grown in the presence of streptomycin (50 mg.mL-1). ocp transcription was induced by addition of 11

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IPTG (0.2 mM), and the cells were incubated overnight at 28°C. The OCP was isolated

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as described in [31]. The FRP overexpression in E. coli cells and its isolation was

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realized using the method described in [26, 30].

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2.3 Holo-OCP concentration calculation

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Total OCP was measured using the Bradford method. At least five independent Bradford measurements of each OCP isolate were carried out. The concentration in

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mg/mL was converted to molar concentration using a MW of 35 kDa for the OCP. Holo-OCP concentration was calculated based in the fact that each holo-OCP binds

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one carotenoid molecule and thus, the molar concentrations of carotenoid and holoOCP are identical. Carotenoid concentration was first calculated in mg/mL from the

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carotenoid absorbance at 496 nm and using A1%1cm = 2158 and then converted to molar concentration. The molar ratio holo-OCP to total OCP gave the percentage of holo-protein. When this ratio was around 1, we considered that the preparation contained 100% holo-protein.

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2.4 PBS isolation

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Synechocystis PBS were isolated as described in [4]. Briefly, after reaching optical density = 1 at 800nm, cyanobacteria cells were pelleted. Cells were broken with glass beads, phycobilisomes solubilized by Triton X-100 (Sigma-Aldrich) treatment and then

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isolated on a sucrose gradient.

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2.5 Absorbance measurements: photoactivation and recovery kinetics

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measurements

Absorbance spectra and the kinetics of photoactivity (illumination with 5000 µmol

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photons m-2 s-1 of white light) and dark recovery were measured in a Specord S600 (Analyticjena) at 8°C or 23°C in 1cm pathlength cuvette. The presence of apo-OCP in the samples could induce a bias since FRP is also able to bind apo-OCP. Thus, total OCP concentration (determined by Bradford) and not holo-protein concentration was used for OCP:FRP ratio calculations. The concentration of apo-protein in each sample was calculated as in [31]. In supplementary Fig 2 is described the effect of the presence of apo-protein on FRP activity. At identical concentrations of holo-protein and FRP, the acceleration of the OCPr to OCPo conversion slows down with increasing 13

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apo-protein concentrations. Nevertheless, the concentration of apo-protein was less

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than 20% in the WT, all simple mutants and the double mutant E261V-D262L. In the

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preparations of double mutants D220K-F299R, D220K-D262K and D220K-N236K the

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were calculated to be respectively 0.5, 1, 4 or 8.

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concentration of apo-protein was around 70% higher. The molar ratios of FRP to OCP

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2.6 Fluorescence measurements: Recovery of PBS fluorescence

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Fluorescence yield quenching and recovery were monitored using a pulse amplitude

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fluorometer (101/102/103-PAM; Walz). Measurements were performed in a 1 cm path-length stirred cuvette. In vitro reconstitution was done as described in [4] with a

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PBS concentration of 0.012µM in 0.5M potassium phosphate buffer (pH=7.5) at 23°C. The OCP was previously converted to the red form by 10 min illumination with strong white light (5000 µmol photons. m-2 s-1) at 4°C. After OCPr addition to the PBS, fluorescence quenching was followed under 1000 µmol photons.m-2s-1 blue-green light (halogen white light filtered by a Corion cut-off 550-nm filter; 400-550nm). After 5 min illumination, the light was turned off and the recovery of fluorescence yield was followed in the absence or presence of FRP (2µM and 16µM final). FRP was added at the same time that the light was turned off. The ratio OCP to PBS was 40 in 14

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all the experiments. The concentration of OCP for these experiments was calculated

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from the carotenoid absorbance spectra since only the OCP attaching a carotenoid

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can be photoactivated and bound to PBS.

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2.7 Coimmunoprecipitation.

Anti-FRP antibodies (Covalab), FRPs and OCP C-terminal domain were incubated at 4

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°C overnight. The Sepharose-protein A beads (Sigma) were blocked by incubating

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them in the presence of 5% BSA overnight. The mixed proteins were then incubated

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with the blocked Sepharose beads for 2.5 h. The beads were washed with a buffer containing 1% β-DM to eliminate non-bound proteins. The proteins attached to the

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anti- FRP antibodies were eluted with 2% SDS buffer and loaded onto an SDS-gel. In the control samples, the FRP was absent. Western blotting was performed using an anti-OCP antibody.

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3. RESULTS

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3.1 Photoactivation and Recovery reactions of OCP mutants:

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To study the interaction between OCP and FRP and identify essential OCP

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amino acids for this interaction we produced recombinant modified holo-OCPs in E. coli cells synthesizing echinenone (ECN) [31]. In these cells, which contain 15-25% of

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-carotene, 70-80% ECN and 4-6% canthaxanthin, OCP was previously shown to

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attach only to ECN [31]. The E. coli strain used for OCP production contained two

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plasmids. The first plasmid carried the operon containing the 4 genes (crtE, crtY, crtI and crtB) from Erwinia herbicola needed for the synthesis of -carotene from its

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precursor, farnesyl-diphosphate; the operon was under the control of the constitutive crtE promoter. The plasmid also carried the Synechocystis crtO gene coding for a -carotene ketolase which is necessary for ECN synthesis; crtO gene was under the control of the arabinose inducible pBAD promoter. The second plasmid carried the modified ocp genes under the control of the T7 promoter (induction with IPTG). A sequential induction of the crtO and ocp genes was essential to obtain a high percentage of holo-OCPs. The crtO gene was first induced in E. coli containing relative high concentrations of -carotene. Subsequently, the ocp gene was induced. The 16

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proteins obtained with this method carried an N-terminal His-tag and simple or

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double specific mutations. The following OCP mutants were constructed and isolated:

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D220K, R229L, N236K, E261K, D262K, F299R, D220K-N236K, D220K-D262K, D220K-

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F299R, E261K-D262K, E261L-D262V (Figure 1). The absorbance spectra of the OCPo form of all these mutated OCPs are identical to those of the WT OCP (Supplementary

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Figure 3). The kinetics of photoactivation (OCPo to OCPr) and recovery (OCPr to OCPo) were followed by measuring optical density changes at 550 nm during 5 min

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illumination with strong white light and then in darkness (Supplementary Figure 4

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and Figure 2).

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Figure 2A and 2B shows that some of mutations have an effect on the stability of OCPr. At 8°C, OCPs containing mutations in E261 and D262 were mostly converted

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(80-90%) to the orange inactive form after 20 min of dark incubation and the R229L OCP recovered 60% (Figure 2). In contrast, the WT and the other mutated OCPrs were very stable at this temperature and recovered less than 25% in this period of time (Figure 2). The fast recovery of OCPs containing mutations in E261 and D262 affected the accumulation of OCPr. Even at 8°C these mutated OCPs were only partially photoconverted to the red form (Supplementary Figure 4).

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3.2 Effect of mutations on FRP interaction with free OCP

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We first followed the effect of FRP on the OCPr to OCPo dark conversion at 8°C. The WT and mutated OCPs were illuminated for 5 min with strong white light and then FRP (0.5 FRP per 1 OCP) was added and the light turned off. Figures 2C and 2D

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show the kinetics of OCPr to OCPo conversion in the presence of FRP. The FRP

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accelerated the recovery of all mutated OCPs with the exception of the F299R and the D220K-F299R OCP mutants. In these two mutants FRP had no effect at all. Even

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detected (Figure 3A).

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when 4 and 8 FRP per OCP were added, no acceleration of the recovery reaction was

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It was also observed that although FRP accelerated the recovery reaction of the D220K simple mutant, this acceleration was much smaller than that observed in the WT (Figure 2C). This was also observed in the double mutants D220K-D262K and D220K-N236K (Figure 2D). The recovery kinetics in the presence of FRP were similar for the WT, the simple mutants E261K, D262K and double mutants E261K-D262K, E261L-D262V. This was also true when 1 or 4 FRPs per OCP were added and there was a direct relationship between the increase of the FRP/OCP ratio and the acceleration of the OCPr to OCPo conversion. These results are described in detail in 18

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Figure 3B for the WT and the E261K OCP mutant as an example. Our results strongly

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suggest that while OCP amino acids R229, N236, E261 and D262 have a minor (or no)

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role in the interaction with FRP, F299 is essential for FRP activity and D220 also has

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important role.

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In order to confirm this hypothesis we tested the effect of the FRP when it was

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added during the illumination period (Figure 4). FRP (1 FRP per 1 OCP) was added before illumination (5 min) with strong white light. Figure 4 compares the kinetics of

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OCPr accumulation in the absence and presence of FRP at 8°C and 23°C. The presence

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of FRP almost completely inhibited the accumulation of OCPr when WT OCP was

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tested. In contrast, addition of FRP to the F299R OCP had no effect on the kinetics. When 8 FRPs per OCP F299R were added also no effect was observed (supplementary

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Figure 5A). In the case of the D220K mutant, FRP decreased the accumulation of OCPr but much less than in the case of the WT OCP. The effect of FRP was larger at 23°C than at 8°C indicating that the interaction OCP-FRP is temperature dependent and relies on the presence of the OCP F299 residue and to some extent on that of the D220 residue. FRP also almost completely inhibited the accumulation of OCPr when E261K and E261K-D262K OCP mutants were tested (supplementary Figure 6).

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3.3 Effect of OCP mutations on FRP interaction with OCP bound to phycobilisomes

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In vitro reconstitution experiments, developed by [4], were used to elucidate the role of the F299 and D220 amino acids in the interaction between the FRP and the OCP bound to PBS. Isolated Synechocystis PBS were incubated with pre-

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illuminated WT and F299R, D220K and F299R-D220K mutated OCPrs, in 0.5M

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phosphate under strong blue-green light at 23°C. After 5 min of illumination during which a decrease of PBS fluorescence was observed in all the cases, the light was

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turn-off and the fluorescence recovery was followed by measurements in a PAM

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fluorometer in the absence or presence of FRP (2µM and 16µM) (Figure 5B and 6).

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Figure 5B shows the fluorescence recovery kinetics in the absence of FRP. In the past we showed that while the WT OCP carrying a C-terminal His-tag remained attached to the PBS for several hours in 0.5M phosphate, the OCP carrying an Nterminal His-tag weakly attached to the PBS at this phosphate concentration and PBS fluorescence was completely recovered after 20 min of dark incubation [31]. This is also observed in Figures 5B and 6A. The F299R mutation had a clear influence on fluorescence recovery kinetics: PBS fluorescence recovery with F299R OCP was extremely slow (only 40% recovery after 20 min). Since the binding of OCP to PBS is 20

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an equilibrium reaction that largely depends on the concentration of OCP r, this could

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be explained by the fact that the F299R OCPr form is accumulated very fast and it is

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more stable than the WT OCPr form (Figures 2 and supplementary Figure 4).

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Moreover, the stabilization of the red form was larger for the F299R mutant than for the WT and other mutants under the conditions used in the fluorescence recovery

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experiment (23°C in the presence of 0.5M phosphate (Figure 5A).

Unexpectedly, the addition of FRP accelerated the recovery of PBS fluorescence

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kinetics in all cases, even when the F299R and D220K-F299R mutants were used

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(Figure 6). FRP had no effect on the OCPr to OCPo dark recovery at 8°C and at 23°C on

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these mutants (Figures 3, 4 and supplementary Fig 4A). In the past, we observed that a large excess of FRP (16 µM) must be added in order to see an effect of FRP on

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fluorescence recovery [4]. In our experiments we used an FRP concentration of 16µM which accelerated the fluorescence recovery by a factor of 2 in all cases (Table I). The acceleration was only slightly lower with the F299R and D220K-F299R mutants. This may be related to a higher concentration of OCPr due a nil effect of the FRP on the OCPr to OCPo recovery. Addition of 2µM FRP also accelerated the fluorescence recovery 1.2 to 1.7-fold when the WT and mutants carrying an N-terminal His-tag were used (Table I). As expected, there was a slightly larger effect of FRP when we tested OCPs having an accelerated OCPr to OCPo conversion in the presence of FRP. 21

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The simple mutant E261K and the double mutant E261V-D262L were also studied

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because they seemed to have a key role in the current model for OCP-FRP interaction

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[28]. The addition of FRP also accelerated the recovery of PBS fluorescence kinetics

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when these mutants were used (supplementary Figure 7 and Table I).

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3.4 Effects of the R60L FRP mutation on OCPr to OCPo conversion and fluorescence

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recovery

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In Sutter et al., 2013, [28] we showed that the R60L FRP mutant had no effect on the

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OCPr to OCPo conversion at 8°C when the ratio of FRP to OCP was 0.5. This was also observed when the ratio of FRP to OCP was equal to 8, at 8°C (Figure 7B) and at 23°C (supplementary Fig 5B). Moreover, the addition of 8 FRP per OCP did not affect the

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accumulation of OCPr during illumination with strong white light (Figure 7A).

By contrast, the addition of the R60L FRP mutant (2µM and 16µM) to PBS-WT OCP quenched complexes accelerated PBS fluorescence recovery (Figure 7C). Hence, although the R60L FRP mutant was unable to accelerate the OCPr to OCPo conversion, it was able to interact with bound OCP and help its detachment from the PBS. In order to confirm the interaction between R60L FRP and OCP, co-immuno 22

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precipitation experiments were carried out. The C-terminal domain of Synechocystis

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OCP produced in Synechocystis cells [28] was used in this experiment because in the

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absence of the N-terminal domain, this interaction is light independent.

The C-terminal domain was incubated with FRP antibodies and the WT and

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mutant FRPs and without any FRP as a control. Then the FRP antibodies were fixed to

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block Sepharose-protein A beads. After washing off the free proteins, the proteins attached to the anti-FRP antibodies were eluted and loaded on an SDS-

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electrophoresis gel. CTD was detected by Western-blot using and anti-OCP antibody.

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Figure 8 clearly shows that the CTD not only co-precipitated with the WT FRP but also

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OCP.

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with the R60L FRP mutant confirming that this mutated FRP can interact with the

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4. DISCUSSION

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4.1 FRP action on free OCP: Acceleration of OCPr to OCPo conversion

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In the Sutter et al. docking model, the FRP dimer binds to one side of the OCP

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C-terminal domain, including the loops connecting the -sheet strands [28]. Several OCP amino acids have been proposed to be involved in this interaction: T218, D220,

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R229, V232, N236, D262, F264, and F299 [28]. These amino acids are partially hidden

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in OCPo by the N-terminal arm, which sticks to the C-terminal domain in the dark

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inactive form. This could explain why FRP preferentially binds OCPr [26, 28, 29] in which the N-terminal arm is detached from the CTD and the CTD and NTD are

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completely separated [12, 14]. In the present work, by constructing several single and double mutants, we have demonstrated that F299 is essential for FRP activity as accelerator of the OCPr to OCPo conversion: when WT FRP was added to F299R OCP, there was no effect on the conversion rate at 8 and 23°C even when the ratio of FRP to OCP was 8:1. While WT OCP photoactivation was almost completely prevented when OCP was illuminated in the presence of FRP (1 FRP per OCP), the kinetics of F299R OCP photoactivation was not modified by the presence of FRP. The action of FRP was also affected when amino acid D220 was modified: although this 24

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modification did not completely prevent the action of the FRP, it largely hindered its

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activity. By contrast, modifications of amino acids R229, N236, E261 and D262 did not

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affect FRP activity. The N-terminal arm, which is a versatile structure, was shown to

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unstick from the CTD upon photoactivation [12, 14, 18]. The CTD region covered in the inactive form (including F299) becomes accessible for FRP binding. FRP could bind

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CTD region where D220 and F299 are located (Figure 1). These two residues are in close vicinity in the 3D structure of OCP (3.8Å on PDB ID: 3MG1 structure): D220 is

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located on β1 and F299 on β5. It was also shown that the C-terminal domain does not rearrange upon photoactivation [12]. Thus, residues at the junction between β1 and

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β5 appear to be crucial for the FRP-OCP-closing activity, not β-sheet rearrangements. Our study has also shown that E261 and D262 which surround the N-terminal arm, as

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well as N236 which is located on the small α-helix connecting β2 and β3, are not

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important for FRP activity on free OCPr (Figure 1).

4.2 FRP action on OCP bound to PBS

The effect of FRP on PBS fluorescence recovery was also studied. Surprisingly, we observed that the presence of FRP accelerated the recovery of lost PBS fluorescence by OCP-PBS complexes in all cases, even when F299R and F299R-D220K mutants were used. The kinetics of PBS fluorescence are influenced by both the 25

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concentration of free OCPr in solution and the strength of OCP binding to PBS.

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Differences in PBS fluorescence recovery were observed even in the absence of FRP:

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recovery was much faster when E261 and D262 OCP mutants were used and very

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slow with F299R OCP. In the first case, the fact that OCPr is very unstable in E261K and E261L-D262V mutants can largely explain the fast fluorescence recovery

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observed but it is not possible to discard the hypothesis of weaker OCP binding to PBS. The red form of the F299R OCP mutant is more stable than that of WT OCP at all

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temperatures and the accumulation of its OCPr is faster. This can partially explain the very slow PBS fluorescence recovery observed in OCP-PBS complexes containing this

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mutated OCP. However, a stronger OCP binding to PBS is most likely to also be contributor to the slow fluorescence recovery. The effect of these mutations on OCP

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binding cannot be explained by the existing models describing OCP binding to PBS [23, 24, 33]. In the most recent model in which NTD is buried between two APC

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trimers and CTD is located on the outer surface of a basal cylinder, these CTD amino acids are oriented toward the opposite side of PBS and have no contact with PBS [23]. Another explanation could be that these mutations affect the structure OCP r and the separation of the CTD and NTD domains, influencing in an indirect way OCP binding to PBS.

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The addition of FRP accelerates fluorescence recovery of all OCP-PBS complexes even

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when the R60L FRP was used. This indicated that WT FRP was able to interact with all

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PBS-bound OCPs including F299R OCP and accelerated their detachment from the

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PBS. Furthermore, R60L FRP, which is inactive in the acceleration of OCPr to OCPo conversion, clearly interacts with bound OCPr. Thus, modification of one (or two) OCP

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or FRP amino acids that are crucial for the action of FRP on the OCPr to OCPo conversion is not sufficient to prevent FRP interaction with the OCP, at least in the

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bound form. This might be explained by three different hypotheses: 1) the binding site of FRP could be different in free and bound OCPr; 2) many amino acids of OCP

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and FRP could be involved in the interaction and modification of one or two of them may not be sufficient for breaking the interaction; 3) different amino acids are

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involved in the two distinct activities of the FRP.

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4.3 FRP has two distinct activities

The results described in this article strongly suggest that FRP has two distinct activities. We infer this from the fact that: 1) a mutated FRP (R60L) unable to accelerate the OCPr to OCPo conversion is able to accelerate the recovery of the lost PBS fluorescence in OCP-PBS complexes; and 2) the WT FRP is unable to accelerate the OCPr to OCPo conversion of the F299R OCP but is able to accelerate the recovery of PBS fluorescence. Thus, FRP has two activities, (1) OCP detachment from the PBS 27

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and (2) its conversion to the orange form. For the second activity, F299 in OCP and

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R60 in FRP are essential. A recent report has suggested that FRP dimers dissociate

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upon binding to OCPr [29]. The authors proposed a model in which FRP binds as a

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dimer to OCPr and then, one monomer is detached and the other monomer brings together the CTD and NTD domains, stabilizing an intermediate state that facilitates

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the OCPr to OCPo conversion [29]. In the dimer, R60 of one monomer is involved in a cation-π interaction with W50 of the other monomer of the FRP dimer. If the

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monomer dissociates, it could be free to forms a cation-π interaction with an OCP amino acid, for example F299. This interaction could be essential for the formation

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and stabilization of the intermediate state. If it is confirmed that the FRP dimer binds to the PBS-bound OCP, but the FRP monomer accelerates the OCPr to OCPo

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conversion of free OCP, we can hypothesize that the site of interaction changes and

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that different amino acids might be involved.

4.4 Mechanism of FRP action

In Figure 9, we propose a model for FRP action: when OCP is bound to PBS, only the C-terminal domain is accessible because the N-terminal domain burrows into the PBS to provide photoprotection [23]. Thus, FRP first binds the OCP C-terminal domain [28] as a dimer causing OCP detachment from PBS. The mechanisms 28

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underlying this process remain unknown. Moreover, for the moment no OCP or FRP

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amino acids have been demonstrated to be essential for the interaction of FRP with

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the bound OCPr.

The binding of the FRP dimer to OCP could cause sufficient steric hindering to

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destabilize the OCP-PBS interaction, and following (or simultaneously to) this dissociation, the monomerization of FRP could occur as proposed by Sluchanko et al.

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[29]. From our work, we infer that a very specific interaction involving at least OCP amino acids F299 and D220 (this work) and FRP amino acids R60, W54 and D50 [28] is

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established once OCPr is detached from the PBS. This interaction by an unknown mechanism facilitates the closure of the OCP and translocation of the ketocarotenoid

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to its position in OCPo. Monomerization of FRP could be crucial to this specific interaction, but needs to be confirmed. In order to reset the system and have fully

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functional OCPs, FRP must be separated from OCP. Closure of the OCP and attachment of the N-terminal arm to the C-terminal arm probably destabilizes the OCP-FRP interaction resulting in the dissociation of the FRP monomer from OCP. The FRP dimer could then reassociate.

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Acknowledgments

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We thank Prof. Gerhard Sandmann for the gift of plasmid pACCAR16crtX. We thank

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Prof Cheryl A Kerfeld for her helping to choose the amino acids to be mutated and for helpful discussions. This work was supported by the Agence Nationale de la

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Recherche (project CYANOPROTECT), the Centre National de la Recherche Scientifique, the Commissariat à l’Energie Atomique, Paris-Saclay University (IDI

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project grant no. ANR–11–IDEX–0003–02 to A.T.) and Phycosource company (salary

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of C.B.C.).

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[33] I.N. Stadnichuk, M.F. Yanyushin, E.G. Maksimov, E.P. Lukashev, S.K. Zharmukhamedov, I.V. Elanskaya, V.Z. Paschenko, Site of non-photochemical

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Legend of Figures

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Figure 1: Structure of the OCP from Synechocystis sp. PCC 6803 (PDB ID: 3MG1;

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[11]). (A) The OCP monomer is represented in the orange state. The N-terminal domain (residues 1–165) is red. The C-terminal domain (residues 196-315) is yellow.

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In purple sticks, residues modified in this study are represented. The carotenoid is represented as orange sticks. (B) Zoom of the FRP putative binding site on OCP C-

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terminal domain.

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Figure 2: Dark recovery kinetics: OCPr to OCPo of isolated wild-type and mutated

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OCPs at 8°C, in the absence (A and B) and presence (C and D) of FRP. A, OCPr to OCPo

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dark recovery of wild-type OCP (close circles, black) and simple mutants OCPs: D220K

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(open squares, green), F299R (open circles, pink), E261K (open triangles, blue), D262K (close squares, purple), D229L (close diamond, grey) and N236K (close triangles,

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orange). B, OCPr to OCPo dark recovery of double mutants OCPs: E261L-D262V (open triangles, blue), E261K-D262K (close triangles, green), D220K-D262K (open circles,

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blue), D220K-F299R (close circles, pink) and D220K-N236K (close squares, purple). C, OCPr to OCPo dark recovery of wild-type OCP and simple mutants OCPs in the

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presence of FRP using the same symbols than in A. D, OCPr to OCPo dark recovery of wild-type OCP and double mutants OCPs in the presence of FRP using the same

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symbols than in B. The lines represent the mean of three independent experiments.

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The error bars represent the SD.

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Figure 3: Effect of FRP on OCP dark recovery kinetics (OCPr to OCPo) at 8°C of : A,

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F299R simple mutant in the absence of FRP (close squares, purple) and in the

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presence of 1 FRP for 1 F299R OCP (close circles, pink), 4 FRP for 1 F299R OCP (open

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circles, blue) and 8 FRP for 1 F299R OCP (open triangles, blue). B, Wild type OCP in the presence of 1 FRP for 1 WT OCP (open triangles, pink) and 4 FRP for 1 WT OCP

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(close circles, blue). E261K simple mutant in the presence of 1 FRP for 1 E261K OCP (open squares, black) and 4 FRP for 1 E261K OCP (close squares, blue). The lines

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represent the mean of three independent experiments. The error bars represent the

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SD.

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Figure 4: Effect of FRP on photoconversion kinetics: OCPo to OCPr. Photoactivation

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kinetics of isolated WT and mutated OCPs at 8°C (A and C) and 23°C (B) in the

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absence (close symbols) or presence of FRP (open symbols). The OCPs were

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illuminated with white light (5000 μmol photons m−2 s−1). A, 8°C, WT OCP in the absence of FRP (close squares, black) and in the presence of FRP (open squares,

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black), F299R without FRP (close triangles, purple) and with FRP (open triangles, purple), and D220K without FRP (close circles, blue) and with FRP (open circle, blue).

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B, 23°C, WT OCP in the absence of FRP (close diamonds, black) and in the presence of FRP (open squares, black), F299R without FRP (close triangles, purple) and with FRP

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(open triangles, purple) and D220K without FRP (close circle, blue) and with FRP (open circle, blue). Each graph shows a representative experiment. The

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measurements were done all in the same day. The experiment was repeated twice.

Figure 5: (A) Dark recovery kinetics (OCPr to OCPo) of isolated WT and mutated OCPs at 23°C in 0.5M phosphate and (B) dark PBS fluorescence recovery in quenched OCP-PB complexes containing WT and mutated OCPs (23°C, 0.5M phosphate). WT OCP (open circles, black), F299R (open triangles, purple), D220K (open squares, pink), and D220K-F299R (close triangles, orange). The results shown in (A) are the mean of three independent experiments and the error bars represent the SD. In (B) a representative experiment is shown. 42

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Figure 6: Effect of FRP on PBS fluorescence recovery kinetics. Dark PBS fluorescence

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recovery in quenched OCP-PB complexes in the absence (red lines) or presence of

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2μM of FRP (blue lines), 16μM of FRP (black lines) of WT OCP (A), D220K (B), F299R

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(C), D220K-F299R (D). The experiments were repeated three times. The error bars

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(SD) as shown by the thickness of lines.

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Figure 7: Effect of FRP R60L mutant on (A) WT OCP photoconversion, (B) WT OCP dark recovery at 8°C and (C) dark PBS fluorescence recovery kinetics at 23°C. (A and

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B) Photoactivation kinetics in the absence of FRP R60L (close circles, red) or in the

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presence of 8 FRP R60L for 1 WT OCP (open circles, blue). Mean of three independent

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experiments. Error bars represent SD. (C) Dark PBS fluorescence recovery in quenched OCP-PB complexes in the absence (red line) or presence of 2μM of FRP

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R60L (blue line), 16μM of FRP R60L (black line) of WT OCP. Mean of three independent experiments. The thickness of lines represents the error (SD).

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Figure 8: Analysis of the FRP-OCP interaction by co-inmuno precipitation. Coomassie

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blue-stained SDS-PAGE (upper panel) and Anti-OCP immunoblot (down panel, black

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dotted frame) of OCP C-terminal domain after co-immunoprecipitation with WT FRP

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(WT) or R60L FRP (R60L). Control experiment without FRP (-FRP) is also shown. In the

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upper panel M lane is the size marker. In the down panel the 20 kDa marker is visible

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Figure 9: Working model for the two distinct activities of FRP. 1. FRP binds to the CTD of the bound OCP. 2. Upon binding, FRP monomerizes and helps the detachment

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of OCPr from the PBS. 3. FRP accelerates the OCPr to OCPo conversion. 4. FRP

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detaches from OCP and dimerizes. For details, see text.

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B

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80

40

60

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F299R D220K D262K E261K N236K WT R229L

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% of OCP red

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20

D220K-F299R D220K-D262K D220K-N236K E261L-D262V E261K-D262K

20

400

600

800

1000

0

1200

0

200

400

NU

Time (sec)

C

600

800

1000

1200

Time (sec)

D +FRP

100

+FRP

100

D220K-F299R

MA

F299R

80

60

D

D220K

TE

40

20

0

200

400

CE P

0 600

Time (sec)

800

1000

1200

% of OCP red

80

60

D220K-D262K 40

20

D220K-N236K

0 0

200

400

600

800

1000

1200

Time (sec)

AC

% of OCP red

T

100

% of OCP red

100

SC R

A

Figure 2: Dark recovery kinetics: OCPr to OCPo of isolated wild-type and mutated OCPs at 8°C, in the absence (A and B) and presence (C and D) of FRP. A, OCPr to OCPo dark recovery of wild-type OCP (close circles, black) and simple mutants OCPs: D220K (open squares, green), F299R (open circles, pink), E261K (open triangles, blue), D262K (close squares, purple), D229L (close diamond, grey) and N236K (close triangles, orange). B, OCPr to OCPo dark recovery of double mutants OCPs: E261L-D262V (open triangles, blue), E261K-D262K (close triangles, green), D220K-D262K (open circles, blue), D220KF299R (close circles, pink) and D220K-N236K (close squares, purple). C, OCPr to OCPo dark recovery of wild-type OCP and simple mutants OCPs in the presence of FRP using the same symbols than in A. D, OCPr to OCPo dark recovery of wild-type OCP and double mutants OCPs in the presence of FRP using the same symbols than in B. The lines represent the mean of three independent experiments. The error bars represent the SD.

Fig 2

46

ACCEPTED MANUSCRIPT

A

100

F299R

T IP

80

70

SC R

% of OCP red

90

No FRP 1 FRP/OCP 4 FRP/OCP 8 FRP/OCP

60

50 0

200

400

600

800

1000

1200

B

WT 4FRP/OCP E261K 4FRP/OCP WT 1FRP/OCP E261K 1FRP/OCP

60

40

D

% of OCP red

80

MA

100

NU

Time (sec)

0 0

50

100

TE

20

150

200

AC

CE P

Time (sec)

Figure 3: Effect of FRP on OCP dark recovery kinetics (OCPr to OCPo) at 8°C of : A, F299R simple mutant in the absence of FRP (close squares, purple) and in the presence of 1 FRP for 1 F299R OCP (close circles, pink), 4 FRP for 1 F299R OCP (open circles, blue) and 8 FRP for 1 F299R OCP (open triangles, blue). B, Wild type OCP in the presence of 1 FRP for 1 WT OCP (open triangles, pink) and 4 FRP for 1 WT OCP (close circles, blue). E261K simple mutant in the presence of 1 FRP for 1 E261K OCP (open squares, black) and 4 FRP for 1 E261K OCP (close squares, blue). The lines represent the mean of three independent experiments. The error bars represent the SD.

Fig 3

47

ACCEPTED MANUSCRIPT

A100

8°C

T

60

WT WT FRP

IP

% of OCPred

80

F299R F299R FRP

40

D220K

SC R

D220K FRP

20

0 0

50

100

150

200

250

300

B100

NU

Time (sec) 23°C

MA

WT WT FRP F299R F299R FRP D220K D220K FRP

60

40

D

% of OCPred

80

0 0

50

100

150

TE

20

200

300

CE P

Time (sec)

250

Figure 4: Effect of FRP on photoconversion kinetics: OCPo to OCPr. Photoactivation kinetics of

AC

isolated WT and mutated OCPs at 8°C (A and C) and 23°C (B) in the absence (close symbols) or presence of FRP (open symbols). The OCPs were illuminated with white light (5000 μmol photons m−2 s−1). A, 8°C, WT OCP in the absence of FRP (close squares, black) and in the presence of FRP (open squares, black), F299R without FRP (close triangles, purple) and with FRP (open triangles, purple), and D220K without FRP (close circles, blue) and with FRP (open circle, blue). B, 23°C, WT OCP in the absence of FRP (close diamonds, black) and in the presence of FRP (open squares, black), F299R without FRP (close triangles, purple) and with FRP (open triangles, purple) and D220K without FRP (close circle, blue) and with FRP (open circle, blue). Each graph shows a representative experiment. The measurements were done all in the same day. The experiment was repeated twice.

Fig 4

48

ACCEPTED MANUSCRIPT

A

23°C 0.5M phosphate

100

IP

40

SC R

% of OCPred

60

T

WT F299R D220K D220K-F299R

80

20

0 0

100

200

300

400

500

600

NU

Time (sec)

B 80

WT F299R D220K D220K F299R

60

D

40

20

0 0

200

400

600

TE

% of initial Fluorescence

MA

Recovery without FRP

100

800

1000

1200

AC

CE P

Time (sec)

Figure 5: (A) Dark recovery kinetics (OCPr to OCPo) of isolated WT and mutated OCPs at 23°C in 0.5M phosphate and (B) dark PBS fluorescence recovery in quenched OCP-PB complexes containing WT and mutated OCPs (23°C, 0.5M phosphate). WT OCP (open circles, black), F299R (open triangles, purple), D220K (open squares, pink), and D220K-F299R (close triangles, orange). The results shown in (A) are the mean of three independent experiments and the error bars represent the SD. In (B) a representative experiment is shown.

Fig 5

49

ACCEPTED MANUSCRIPT

40

20

60

40

20

0

0 0

200

400

600

800

1000

0

1200

Time (sec)

C 100

200

400

600

800

1000

1200

1000

1200

Time (sec)

D

NU

F299R

D220K-F299R

80

MA

60

% of initial Fluorescence

100

80

40

0 0

200

400

600

800

TE

D

20

1000 1200 1400

40

20

0 0

200

400

600

800

Time (sec)

CE P

Time (sec)

60

AC

% of initial Fluorescence

T

60

80

IP

80

D220K

100

% of initial Fluorescence

% of initial Fluorescence

B

WT

100

SC R

A

Figure 6: Effect of FRP on PBS fluorescence recovery kinetics. Dark PBS fluorescence recovery in quenched OCP-PB complexes in the absence (red lines) or presence of 2μM of FRP (blue lines), 16μM of FRP (black lines) of WT OCP (A), D220K (B), F299R (C), D220KF299R (D). The experiments were repeated three times. The error bars (SD) as shown by the thickness of lines.

Fig 6

50

ACCEPTED MANUSCRIPT

FRP R60L 8°C

90

0.08 0.06 0.04

80 70 60 50

A

0 0

50

100

150

200

250

30 300

60

40

0

200

400

600

800

1000

Time (sec)

B1200

C

0

0

200

400

600

800

1000 1200

Time (sec)

NU

Time (sec)

20

SC R

40

0.02

80

IP

% of OCP red

0.1

R60L

100

MA

Figure 7: Effect of FRP R60L mutant on (A) WT OCP photoconversion, (B) WT OCP dark recovery at 8°C and (C) dark PBS fluorescence recovery kinetics at 23°C. (A and B) Photoactivation kinetics in the absence of FRP R60L (close circles, red) or in the presence

D

of 8 FRP R60L for 1 WT OCP (open circles, blue). Mean of three independent

TE

experiments. Error bars represent SD. (C) Dark PBS fluorescence recovery in quenched OCP-PB complexes in the absence (red line) or presence of 2μM of FRP R60L (blue line),

CE P

16μM of FRP R60L (black line) of WT OCP. Mean of three independent experiments. The thickness of lines represents the error (SD).

AC

Absorbance 550 nm

8°C

T

100

FRP R60L 0.12

% of initial Fluorescence

0.14

Fig 7

51

ACCEPTED MANUSCRIPT

D

MA

NU

SC R

IP

T

M -FRP WT R60L

TE

Figure 8: Analysis of the FRP-OCP interaction by co-inmuno precipitation. Coomassie bluestained SDS-PAGE (upper panel) and Anti-OCP immunoblot (down panel, black dotted

CE P

frame) of OCP C-terminal domain after co-immunoprecipitation with WT FRP (WT) or R60L FRP (R60L). Control experiment without FRP (-FRP) is also shown. In the upper panel M lane

AC

is the size marker. In the down panel the 20 kDa marker is visible

Figure 8

52

AC

CE P

TE

D

MA

NU

SC R

IP

T

ACCEPTED MANUSCRIPT

53

ACCEPTED MANUSCRIPT

Table I: Time (in seconds) necessary to recover 50% of PBS fluorescence in the absence or presence of FRP. 2µM FRP

16µM FRP

WT

340

200 (1.7)

D220K

280

210 (1.3)

F299R

1300

1050 (1.23)

750 (1.73)

D220K-F299R

295

250 (1.18)

185 (1.59)

E261K

80

50 (1.6)

45 (1.8)

E261L-D262V

80

FRP R60L –WT OCP

340

IP

T

0

MA

NU

SC R

160 (2.1)

120 (2.08)

37 (2.16)

260 (1.31)

180 (1.9)

AC

CE P

TE

D

50 (1.6)

54