Correlated fluorescence quenching and topographic mapping of Light-Harvesting Complex II within surface-assembled aggregates and lipid bilayers

Abstract

Light-Harvesting Complex II (LHCII) is a chlorophyll-protein antenna complex that efficiently absorbs solar energy and transfers electronic excited states to photosystems I and II. Under excess light intensity LHCII can adopt a photoprotective state in which excitation energy is safely dissipated as heat, a process known as Non-Photochemical Quenching (NPQ). In vivo NPQ is triggered by combinatorial factors including transmembrane ΔpH, PsbS protein and LHCII-bound zeaxanthin, leading to dramatically shortened LHCII fluorescence lifetimes. In vitro, LHCII in detergent solution or in proteoliposomes can reversibly adopt an NPQ-like state, via manipulation of detergent/protein ratio, lipid/protein ratio, pH or pressure. Previous spectroscopic investigations revealed changes in exciton dynamics and protein conformation that accompany quenching, however, LHCII-LHCII interactions have not been extensively studied. Here, we correlated fluorescence lifetime imaging microscopy (FLIM) and atomic force microscopy (AFM) of trimeric LHCII adsorbed to mica substrates and manipulated the environment to cause varying degrees of quenching. AFM showed that LHCII self-assembled onto mica forming 2D-aggregates (25–150 nm width). FLIM determined that LHCII in these aggregates were in a quenched state, with much lower fluorescence lifetimes (~0.25 ns) compared to free LHCII in solution (2.2–3.9 ns). LHCII-LHCII interactions were disrupted by thylakoid lipids or phospholipids, leading to intermediate fluorescent lifetimes (0.6–0.9 ns). To our knowledge, this is the first in vitro correlation of nanoscale membrane imaging with LHCII quenching. Our findings suggest that lipids could play a key role in modulating the extent of LHCII-LHCII interactions within the thylakoid membrane and so the propensity for NPQ activation.

Proteoliposomes as energy transferring nanomaterials: enhancing the spectral range of light-harvesting proteins using lipid-linked chromophores

“…Broadening of the peaks increases with decreasing LHCII concentration to a maximum of 19% of the peak area compared to isolated LHCII, suggest that up to 19% of chlorophylls may have an altered environment. Whilst not ideal this is entirely in line with multiple other proteoliposome studies (Adams et al, 2018;Natali et al, 2016;Pandit et al, 2011)where LHCII appears to be destabilized when the protein-to-lipid ratio is very low. We speculate that LHCII-LHCII interactions may be stabilizing.…”

“…Trimeric LHCII complexes from spinach were biochemically purified as described previously (Adams et al, 2018). Briefly, spinach leaves (purchased from a local supermarket) were macerated in ice-cold buffer A (300 mM sucrose, 5 mM EDTA, 50 mM HEPES, pH 7.5), the liquid filtered through muslin cloth and chloroplasts collected by centrifugation, resuspended in buffer B (5 mM EDTA, 10 mM Tricine pH 7.4) and osmotically lyzed by adding an equal volume of buffer C (400 mM sucrose, 5 mM EDTA, 10 mM Tricine, pH 7.4).…”

Proteoliposomes as energy transferring nanomaterials: enhancing the spectral range of light-harvesting proteins using lipid-linked chromophores

“…Our LHCII sample had the expected absorption spectrum with peaks representing chlorophyll (Chl) and carotenoids between 400-500 nm and the Qy bands of Chl b and Chl a at 650 and 675nm, respectively (Figure 1A, green solid). 30 A single fluorescence emission peak at ~681.5 nm represents emission from lowest energy Chl a indicating the highly-connected chlorophyll network expected within trimeric LHCII (Figure 1A, green dotted). 30 The absorption of Texas Red is quite broad with a maximum at 591 nm (Figure 1A, solid red), fitting well into the 'green gap' of LHCII.…”

“…30 A single fluorescence emission peak at ~681.5 nm represents emission from lowest energy Chl a indicating the highly-connected chlorophyll network expected within trimeric LHCII (Figure 1A, green dotted). 30 The absorption of Texas Red is quite broad with a maximum at 591 nm (Figure 1A, solid red), fitting well into the 'green gap' of LHCII. The TR fluorescence emission peak at 610 nm has extensive overlap with the LHCII Chl b absorption (Figure 1A, dotted red), thus the excited state of TR is energetically close to chromophores within the intended acceptor LHCII.…”

“…LHCII protein was extracted from spinach leaves and biochemically purified using the detergent α-DDM, as previously described. 30 Denaturing and native gel electrophoresis indicate a high protein purity and the natural trimeric state (see Figure S1 in the Supporting Information). We chose the small organic chromophore Texas Red (TR) 32 as an ideal candidate for an energy donor to LHCII in membranes because of its complementary spectral properties and its amenability to assembly into lipid bilayers, using a form which is tethered to a DHPE lipid headgroup (TR-DHPE, as purchased).…”