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

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.Oxygenic photosynthesis supports most life on Earth through the absorption of solar energy, which powers the extraction of electrons from water and the subsequent use of those electrons to convert CO₂ into organic compounds (Nelson and Ben-Shem, 2004; Merchant and Sawaya, 2005; Nelson, 2011). The light-dependent reactions of photosynthesis occur within photosynthetic or thylakoid membranes and are catalyzed by two reaction centers, PSI and PSII. Both photosystems have associated light-harvesting complexes (LHCI and LHCII) that act as antenna to efficiently capture light energy. The oxygen-evolving complex (OEC) is an integral component of PSII, catalyzing the extraction of electrons from water. The two photosystems are connected through an intersystem electron transport chain that includes the hydrophobic electron carrier plastoquinone, the membrane-bound cytochrome b₆f complex (cyt b₆f), and the mobile electron carrier plastocyanin. The electrochemical gradient generated during light-driven electron flow is used in the synthesis of ATP by the ATP synthase complex. Components of the photosynthetic apparatus vary among photosynthetic organisms and under different environmental conditions, especially for proteins associated with light-harvesting complexes (Liu and Scheuring, 2013). However, investigations of the mechanisms associated with the dynamic acclimation of photosynthetic electron transport and light harvesting to environmental cues require real-time observations that are difficult to achieve because of limitations in our ability to view such changes (e.g. difficulties in tagging proteins with fluorophores and resolving fluorescent images; Zaks et al., 2013).In vascular plants, thylakoid membranes form a network of interconnected tubular structures enclosing a lumenal space. This membrane system can be divided into two morphologically distinct regions: the grana, which are formed by stacks of appressed membranes; and the stroma lamellae, which are unappressed membranes that form connections between grana stacks. These distinct thylakoid regions are enriched for specific photosynthetic complexes. The major complexes in grana are PSII and LHCII, which can interact and form a variety of PSII-LHCII supercomplexes (Dekker and Boekema, 2005; Kouřil et al., 2012), as well as cyt b₆f (Johnson et al., 2014). Grana stacks are also the site of water oxidation and oxygen evolution; the Mn₄CaO₅ cluster is the PSII cofactor that catalyzes this process (Umena et al., 2011). This cluster resides between the transmembrane subunits of the PSII core (formed by PSII proteins D1 and D2 and their associated pigment cofactors, with PSII reaction center proteins CP43 and CP47, α- and β-subunits of cytochrome b559, and PSII reaction center protein I [PsbI]) and the lumenal, peripheral membrane proteins of the OEC. The OEC is composed of extrinsic membrane polypeptides of 33 kD, 23 kD, and 17 kD, designated oxygen-evolving enhancer protein 1, 2, and 3 (PsbO, PsbP, and PsbQ) that protrude into the thylakoid lumen in vascular and nonvascular plants as well as in green algae. Cyanobacteria also have PsbO, with PsbV and PsbU serving as functional analogs of plant PsbP and PsbQ, respectively (Dekker and Boekema, 2005). Based on removal/reconstitution experiments, these subunits have been shown to be critical for PSII stability and oxygen evolution activity (Kuwabara and Murata, 1983; Ljungberg et al., 1983; Ghanotakis et al., 1984). They may also impact the association of Ca²⁺ and Cl⁻ with PSII, the polypeptide conformation around the manganese cluster, and the formation of channels within PSII that allow access of water to the catalytic site and the exit of protons from the complex as water is oxidized (Bricker et al., 2012).The x-ray crystal structures of purified PsbP (Kohoutová et al., 2009) and PsbQ (Balsera et al., 2005) from spinach (Spinacia oleracea) have been determined. For cyanobacteria, PSII crystals have been used to establish high-resolution structures for PsbO, PsbV, and PsbU (Umena et al., 2011), with more recent analyses at room temperature (Kern et al., 2013). To study the bound state of these peripheral proteins in spinach, electron density maps were established based on cryo-electron microscopy (cryo-EM) and single particle analysis of purified PSII-LHCII supercomplexes, with structural verification based on the removal of extrinsic polypeptides of the complexes from the membranes (Nield et al., 2002). In these structures, determined at less than 2 nm (or 20 Å) resolution, the PsbP and PsbQ subunits of OEC were assigned to a single membrane protrusion, with a second membrane protrusion assigned to PsbO (Boekema et al., 2000a); these topological structures are the most prominent protruding features of the reaction center-containing membrane protein complexes. Since two PSII reaction centers associate to form a dimeric PSII-LHCII supercomplex (Bumba and Vácha, 2003), the six OEC subunits (two PsbO, two PsbP, and two PsbQ) are visualized as four protrusions associated with each supercomplex. However, after the cyanobacterial PSII core structure was solved (including the positions of the extrinsic subunits) and aligned to the cryo-EM of PSII-LHCII supercomplexes (Nield and Barber, 2006), the structure was reevaluated. The PSII lumenal small protruding mass was assigned to the large extrinsic loop of CP47 (encoded by psbB), while the larger protrusion was assigned to PsbO, PsbP, PsbQ, and the large extrinsic loop associated with CP43 (encoded by psbC).Attempts have been made to visualize PSII complexes and proteins in their native membrane environment using transmission electron microscopy (TEM) in conjunction with freeze fracture (Johnson et al., 2011) and negative staining of grana membranes from both spinach (Boekema et al., 2000b) and Arabidopsis (Arabidopsis thaliana; Betterle et al., 2009; Wientjes et al., 2013). These techniques provide little information regarding the extent of the protrusion of the polypeptide subunits out of the plane of the membranes. Even though cryo-electron tomography of isolated chloroplasts and plunge-frozen thylakoid membranes (Daum et al., 2010) and grana stacks (Kouřil et al., 2011) can preserve sample hydration using a vitrification process during freezing, it is difficult to determine the height of the extrinsic thylakoid protein protrusions from the membrane surface. Furthermore, at the resolution obtained with these techniques, the small and large protrusions of each PSII monomer may appear merged into a single structure. This would result in the visualization of only two distinguishable topological entities for each PSII-OEC dimer.The generation of images by atomic force microscopy (AFM), which involves raster scanning by a sharp tip that is in contact with the sample, complements other structural determination methods. The vertical position of the tip is controlled in order to maintain a constant imaging force (balancing interaction forces between the tip and the scanned structure). Control is implemented by a feedback loop that continuously monitors the force with a highly sensitive force sensor that activates a high-precision actuator. Logging the vertical position of the piezoelectric actuator that controls the vertical position of the tip can provide particle height relative to the membrane at high vertical resolution; this is done concurrently with logging the lateral position of each pixel to generate the image (Bippes and Muller, 2011). Probing samples with AFM in air has been employed to image spinach grana membranes (Kirchhoff et al., 2008) to elucidate the arrangement of Arabidopsis PSII-LHCII supercomplexes associated with nonphotochemical quenching (Onoa et al., 2014) and to determine the areal density of Arabidopsis PSII-OEC during the PSII repair cycle (Puthiyaveetil et al., 2014).Most AFM studies of grana have been performed with membrane surfaces exposed to air. This raises issues concerning the extent to which membrane properties are altered during measurements in a nonaqueous environment (Zaks et al., 2013), where it may be impossible to maintain appropriate hydration and ionic conditions. However, AFM has also been used in aqueous medium to establish high-resolution topography images of membrane proteins (Bippes and Muller, 2011) and specifically to characterize the PSII-OEC, which was previously observed as an ordered array within spinach grana membranes (Sznee et al., 2011). In recent studies, a map of the lumenal surface of grana membranes was generated in aqueous medium that distinguishes cyt b₆f dimers from PSII-OEC (Johnson et al., 2014). However, the potential of AFM imaging in a liquid environment has not been realized for the high-resolution analysis of features associated with thylakoid membranes and the PSII-OEC dimer. We used contact mode atomic force microscopy (CM-AFM) to (1) image PSII-OEC topology in liquid medium at high resolution, (2) identify other features/particles associated with grana membranes, and (3) optimize the use of AFM for monitoring the dynamics of thylakoid membrane complexes as the conditions of the environment are modulated (e.g. light, specific ions, and temperature).