Single-molecule trapping and spectroscopy reveals photophysical heterogeneity of phycobilisomes quenched by Orange Carotenoid Protein

Allison H Squires,Peter D Dahlberg,Haijun Liu,Nikki Cecil M Magdaong,W E Moerner,Robert E Blankenship

doi:10.1038/s41467-019-09084-2

Allison H Squires, Peter D Dahlberg + Show 4 more

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https://doi.org/10.1038/s41467-019-09084-2

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The Orange Carotenoid Protein (OCP) is a cytosolic photosensor that is responsible for non-photochemical quenching (NPQ) of the light-harvesting process in most cyanobacteria. Upon photoactivation by blue-green light, OCP binds to the phycobilisome antenna complex, providing an excitonic trap to thermally dissipate excess energy. At present, both the binding site and NPQ mechanism of OCP are unknown. Using an Anti-Brownian ELectrokinetic (ABEL) trap, we isolate single phycobilisomes in free solution, both in the presence and absence of activated OCP, to directly determine the photophysics and heterogeneity of OCP-quenched phycobilisomes. Surprisingly, we observe two distinct OCP-quenched states, with lifetimes 0.09 ns (6% of unquenched brightness) and 0.21 ns (11% brightness). Photon-by-photon Monte Carlo simulations of exciton transfer through the phycobilisome suggest that the observed quenched states are kinetically consistent with either two or one bound OCPs, respectively, underscoring an additional mechanism for excitation control in this key photosynthetic unit.

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The Orange Carotenoid Protein (OCP) is a cytosolic photosensor that is responsible for nonphotochemical quenching (NPQ) of the light-harvesting process in most cyanobacteria
Cytosolic OCP in its inactive orange form (OCPO) noncovalently binds a ketocarotenoid, 3ʹ hydroxy-echinenone (3ʹhECN12,20,21), that bridges the α-helical N-terminal domain (NTD) and the α/β C-terminal domain (CTD), which are joined by a flexible inter-domain linker (Fig. 1a)[12,22]
Initial in vitro studies indicated that the PB is quenched by one OCP29 that binds to the core[8,29,30,31,32,33], and that the NTD of OCP is the module responsible for both binding and quenching PBs34–36

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The Orange Carotenoid Protein (OCP) is a cytosolic photosensor that is responsible for nonphotochemical quenching (NPQ) of the light-harvesting process in most cyanobacteria. Simulated brightness and fluorescence lifetimes for CBPB with different combinations of possible OCP quenching sites, produced using a multi-compartment model of the phycobilisome from the literature[61] that was based on ultrafast transient absorption and emission experiments[31,32,33], closely reproduce the experimentally observed populations only under the key assumption that the two quenched populations are the result of one (state Q1) and two (state Q2) OCPs bound to the PB, respectively.

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