Localization of the CyanoP binding site on photosystem II by surface plasmon resonance spectroscopy

Abstract

Photosystem II (PSII), a large multi subunit membrane protein complex localized in the thylakoid membrane of cyanobacteria and chloroplasts, is the only known enzyme that catalyzes the light-driven oxidation of water. In addition to the membrane intrinsic part of PSII, efficient oxygen evolution requires soluble protein subunits at its luminal interface. In contrast to the detailed crystal structure of the active cyanobacterial complex the characterization of intermediate PSII species related to its assembly and repair is hampered by their instability or low abundance. As most structural variations of the corresponding PSII species are based on a different set of protein factors bound to the luminal interface of the complex we developed a system for interaction analysis between PSII and its soluble interaction partners based on surface plasmon resonance (SPR) spectroscopy. The assay was validated by the correct localization of the extrinsic PSII proteins PsbO, PsbV, and PsbU on the luminal PSII surface and used to determine the unknown binding position of CyanoP, the cyanobacterial homolog of higher plant PsbP. The CyanoP binding site was clearly localized in the center of PSII at a position, which is occupied by the PsbO subunit in mature PSII complexes. Consistently, we demonstrate selective binding of CyanoP to an inactive PSII assembly intermediate that lacks the extrinsic subunits PsbO, PsbV, and PsbU. These findings suggest, that CyanoP functions in the dynamic lifecycle of PSII, possibly in the association of CP47 and CP43 or in photoactivation of the oxygen-evolving complex.

How to build a water-splitting machine: structural insights into photosystem II assembly

“…Next, the amplified DNA was cloned into pIVEX2.3d using NdeI/SmaI to obtain pIVEXPsb28His. Expression and purification of 15 N-labelled Psb28 was carried out as described previously (Cormann et al, 2014) with certain adaptations. Overnight starter cultures were grown on agar plates at 37 °C, supplemented with 1 % (w/v) glucose and 100 µg/ml ampicillin.…”

“…Cultures were incubated at 37 °C under vigorous shaking and at an OD600 of 0.6, and expression was induced with addition of isopropylthiogalactoside to a final concentration of 0.5 mM. After overnight incubation the 15 N-labbled Psb28 was isolated and purified as described for his-tagged CyanoP (Cormann et al, 2014). The purity and integrity of the protein samples were checked by SDS-PAGE (data not shown).…”

Structural Coupling of Extrinsic Proteins with the Oxygen-Evolving Center in Photosystem II

“…Binding of multiple CyanoQ copies to the PSII assembly intermediates without PsbU and PsbV was recently reported ( Liu et al, 2015 ). Similarly, interaction of CyanoP with the binding site in PSII, which is occupied by the PsbO subunit in mature PSII complexes, was suggested ( Cormann et al, 2014 ). These facts suggest that localizations of CyanoP and CyanoQ in cyanobacterial PSII would be largely different form those of PsbP and PsbQ in higher plant PSII.…”

Localization and functional characterization of the extrinsic subunits of photosystem II: an update

“…43) However, a recent study using surface plasmon resonance spectroscopy has indicated that the CyanoP binding site is in the PSII center, which Extrinsic subunits of photosystem II 3 is occupied by the PsbO subunit in mature PSII complexes. 44) It is therefore more likely that CyanoP functions in the PSII assembly process, possibly in the association of CP47 and CP43. The above facts suggest that mature cyanobacterial PSII has four extrinsic proteins: PsbO, U, V, and CyanoQ.…”