Abstract
Natural photosynthetic "thylakoid" membranes found in green plants contain a large network of light-harvesting (LH) protein complexes. Rearrangement of this photosynthetic machinery, laterally within stacked membranes called "grana", alters protein-protein interactions leading to changes in the energy balance within the system. Preparation of an experimentally accessible model system that allows the detailed investigation of these complex interactions can be achieved by interfacing thylakoid membranes and synthetic lipids into a template comprised of polymerized lipids in a 2D microarray pattern on glass surfaces. This paper uses this system to interrogate the behavior of LH proteins at the micro- and nanoscale and assesses the efficacy of this model. A combination of fluorescence lifetime imaging and atomic force microscopy reveals the differences in photophysical state and lateral organization between native thylakoid and hybrid membranes, the mechanism of LH protein incorporation into the developing hybrid membranes, and the nanoscale structure of the system. The resulting model system within each corral is a high-quality supported lipid bilayer that incorporates laterally mobile LH proteins. Photosynthetic activity is assessed in the hybrid membranes versus proteoliposomes, revealing that commonly used photochemical assays to test the electron transfer activity of photosystem II may actually produce false-positive results.
Highlights
Taking a closer look at the LHCII absorption spectrum, there is a clear peak at precisely 675 nm and a lower intensity peak at precisely 650 nm, representing Chl a and Chl b Qy transitions, respectively
The ratio of peak heights between Chl a and Chl b is as expected for LHCII and the subtle shoulder at 475 nm is indicative of the trimeric form of LHCII
The well-established features of plant thylakoids, are explained by the following: (i) PSI and its antenna has maximal Chl a absorption at ~682 nm as compared to the PSII at a maximum of ~677 nm, explaining why this peak is found at longer wavelength compared to isolated LHCII alone, (ii) the PSI peak is known to extend further into the red due to its more numerous low-energy chlorophylls, explaining the observed broadening of the peak towards the red end of the spectrum, (iii) PSII has much lesser Chl b than LHCII, and PSI has even less again, explaining the reduced peak at ~650 nm
Summary
Average fluorescence intensity measured in FLIM of hybrid membranes, as total counts within one corral per frame This vale is found from careful analysis of the corrals from many images of hybrid membranes similar to those shown in Figure S4 (N = 16 corrals); NLHCII/corral, is the estimated number of LHCII trimers per corral, N = Fcorral / FLHCII; Density (LHCII/corral) = NLHCII/corral / Acorral (where each corral has area Acorral = 400 μm2); ALHCII, is the area occupied by a single trimeric LHCII protein complex, as estimated in Table S4; Aprotein(%), estimated surface area fraction of the corral occupied by LHC and PS proteins: 8. LH and PS protein do not transfer vertically from thylakoid membranes into pre-existing DOPC supported lipid bilayers
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