Reconstitution and AFM Observation of Photosynthetic Membrane Protein Assembly in Planar Lipid Bilayers

Abstract

In purple photosynthetic bacteria, light-harvesting complex 2 (LH2) and the light harvesting-reaction center complex (LH1-RC) play the key roles of capturing and transferring light energy and subsequent charge separation. These photosynthetic apparatuses form a molecular assembly; however, how the assembly influences the efficiency of energy conversion is not yet clear. To address this issue, direct observation of the assembly at the molecular level is necessary to analyze its function. In this study, we reconstituted photosynthetic membrane proteins into artificial lipid bilayers and directly observed their assembly by AFM. The absorption spectra of the reconstituted proteins showed characteristic Qy bands of bacteriochlorophyll a that were identical to those of intact proteins. AFM observation of the reconstituted membranes revealed that LH2 and LH1-RC were successfully assembled into the lipid bilayer, and their observed structures were in good agreement with corresponding crystallographic structures. Specifically, binary proteins, i.e., LH2/LH1-RC and LH2/LH1, which form a densely packed molecular assembly, could be clearly identified at the molecular level by this method of observation. Energy transfer from LH2 to LH1-RC in a reconstituted lipid bilayer was observed by steady-state fluorescence spectroscopy. Enhanced energy transfer was confirmed in the membrane phase compared to that in a homogeneous micellar solution. Such reconstituted molecular assemblies are useful experimental platforms to investigate the relationship between supramolecular arrays and function.

Electron Conduction and Photocurrent Generation of a Light-Harvesting/Reaction Center Core Complex in Lipid Membrane Environments

“…We found that the number of H-subunits decreased after several imaging scans, indicating that the H-subunits are pulled out by the AFM tip, as previously described. , Considering this loss of H-subunits, LH1-RC complexes in the reconstituted membrane are predominantly oriented toward the H-subunit side (cytoplasmic side), as depicted in Figure B. This is consistent with our previous reports that showed a uniform orientation of LH1-RC complexes in a lipid membrane. , Figure C shows an AFM image of reconstituted LH1-RC under ambient air conditions (similar to that of C-AFM measurement). Patches of the LH1-RC-reconstituted membrane with a thickness of ∼7 nm were observed (height profile (ii)).…”

“…AFM Observation of LH1-RC in Reconstituted Membranes . LH1-RC reconstituted in the lipid bilayer was observed by AFM (Picoplus5500  Molecular Imaging) using AAC mode at ambient temperature. − The reconstituted membrane solution was placed on a freshly cleaved mica surface, followed by the addition of an MgCl 2 solution; the membranes (either lipid/the LH1-RC core complex = 35 or 50) were absorbed onto mica surface via Mg 2+ . After ∼1 h of incubation, the solution was gently replaced with Milli-Q water, followed by water removal under a gentle stream of air to yield planar membranes.…”

Gating-Associated Clustering–Dispersion Dynamics of the KcsA Potassium Channel in a Lipid Membrane

“…Liposome Extension on the Mica Surface and AFM Observation . Flat lipid bilayers on a mica surface were prepared using the vesicle fusion method. − The proteoliposomal suspension was placed on a mica substrate with a MgCl 2 solution. After ∼15 min of incubation, the solution was gently replaced with buffer at a pH of 4.0 (10 mM succinic acid pH 4.0, 300 mM KCl) or pH 7.5 (10 mM HEPES pH 7.5, 300 mM KCl).…”

The Open Gate Structure of the Membrane-Embedded KcsA Potassium Channel Viewed From the Cytoplasmic Side

“…Flat lipid bilayers on a mica surface were prepered with vesicle fusion method3940. The proteoliposomal suspension was put onto a mica substrate after the addition of MgCl 2 solution.…”