The Use of Contact Mode Atomic Force Microscopy in Aqueous Medium for Structural Analysis of Spinach Photosynthetic Complexes

Abstract

To investigate the dynamics of photosynthetic pigment-protein complexes in vascular plants at high resolution in an aqueous environment, membrane-protruding oxygen-evolving complexes (OECs) associated with photosystem II (PSII) on spinach (Spinacia oleracea) grana membranes were examined using contact mode atomic force microscopy. This study represents, to our knowledge, the first use of atomic force microscopy to distinguish the putative large extrinsic loop of Photosystem II CP47 reaction center protein (CP47) from the putative oxygen-evolving enhancer proteins 1, 2, and 3 (PsbO, PsbP, and PsbQ) and large extrinsic loop of Photosystem II CP43 reaction center protein (CP43) in the PSII-OEC extrinsic domains of grana membranes under conditions resulting in the disordered arrangement of PSII-OEC particles. Moreover, we observed uncharacterized membrane particles that, based on their physical characteristics and electrophoretic analysis of the polypeptides associated with the grana samples, are hypothesized to be a domain of photosystem I that protrudes from the stromal face of single thylakoid bilayers. Our results are interpreted in the context of the results of others that were obtained using cryo-electron microscopy (and single particle analysis), negative staining and freeze-fracture electron microscopy, as well as previous atomic force microscopy studies.

Modelling the role of LHCII-LHCII, PSII-LHCII and PSI-LHCII interactions in state transitions

“…The trimodal nearest-neighbour distribution of PSII in SI (Fig. 3G, blue arrows) is in agreement with the organization of PSII observed in AFM images of grana membranes in SI (7, 43, 65) as is the more disordered arrangement in SII (23, 56). Despite the increase in the number of LHCII and PSI-LHCII complexes in the stromal lamellae in SII compared to SI, PSI nearest-neighbour distributions were indistinguishable (Fig.…”

“…Given the unanswered questions that remain on the precise mechanism of state transitions new approaches are needed to clarify how the altered balance of forces upon LHCII phosphorylation affect the thylakoid macro-organization. The structural model of the thylakoid membrane at the individual protein complex level presented here was made possible by recent breakthroughs in elucidating thylakoid organization and dynamics using in atomic force microscopy (AFM) (7, 23, 43) and structured illumination microscopy (SIM) (31, 44), and publication of high-resolution structures of the key protein complexes involved in the light reactions (45–50). Recently developed algorithms, which made the calculation of whether two or more particles spatially overlap tractable, were also essential (see methods).…”

Lateral Segregation of Photosystem I in Cyanobacterial Thylakoids

“…These analyses showed that the neighbors of PSI are likely to be PSII, as depicted in Figure 5D. The pronounced topology of the PSII water-oxidizing complex on the lumenal side of the membrane aids its identification in AFM topographs of plant thylakoids (Sznee et al, 2011;Johnson et al, 2014;Kirchhoff et al, 2008;Phuthong et al, 2015), but in our measurements, this side of the membrane adheres to the mica support and is not available for imaging. Figure 6 shows that, using a different method to immobilize membranes, PSII complexes can be imaged at low resolution, and they likely sit alongside PSI complexes.…”

Visualizing the Photosynthetic Membrane Proteins in Situ: Atomic Force Microscopy

“…However, their structural state in the native membrane environment and their collective dynamic rearrangements reflecting the physiologically crucial processes of acclimation and adaptation of the photosynthetic apparatus to environmental factors have only recently started to emerge. Atomic force microscopy (AFM) of spinach (Spinacia oleracea) membranes enriched in PSII, reported here by Phuthong et al (2015), is currently one of only a few studies that aims at visualizing the photosynthetic membrane proteins at high resolution (a few nanometers) in an environment close to the native one: unfrozen and aquatic (Sznee et al, 2011;Johnson et al, 2014). Unlike freeze-fracture, negative staining, or cryoelectron microscopy, AFM possesses an enormous potential for the visualization of selected membrane components using the principles of natural proteincofactor or protein-protein interactions (Johnson et al, 2014).…”

“…Unlike freeze-fracture, negative staining, or cryoelectron microscopy, AFM possesses an enormous potential for the visualization of selected membrane components using the principles of natural proteincofactor or protein-protein interactions (Johnson et al, 2014). Phuthong et al (2015) report a high-resolution topography of the lumen-protruding domains of key components of the PSII reaction center: inner antenna chlorophyll proteins, CP43 and CP47, and extrinsic subunits, PsbO/PsbP/PsbQ, involved in oxygen evolution, offering a great potential for monitoring the conformational changes associated with oxygen evolution and related adaptive changes in the complex. In addition, the authors identified a new type of particle protruding on the stromal site of the membrane that is likely to be granal PSI.…”