The Ring Structure and Organization of Light Harvesting 2 Complexes in a Reconstituted Lipid Bilayer, Resolved by Atomic Force Microscopy

Abstract

The main function of the transmembrane light-harvesting complexes in photosynthetic organisms is the absorption of a light quantum and its subsequent rapid transfer to a reaction center where a charge separation occurs. A combination of freeze-thaw and dialysis methods were used to reconstitute the detergent-solubilized Light Harvesting 2 complex (LH2) of the purple bacterium Rhodopseudomonas acidophila strain 10050 into preformed egg phosphatidylcholine liposomes, without the need for extra chemical agents. The LH2-containing liposomes opened up to a flat bilayer, which were imaged with tapping and contact mode atomic force microscopy under ambient and physiological conditions, respectively. The LH2 complexes were packed in quasicrystalline domains. The endoplasmic and periplasmic sides of the LH2 complexes could be distinguished by the difference in height of the protrusions from the lipid bilayer. The results indicate that the complexes entered in intact liposomes. In addition, it was observed that the most hydrophilic side, the periplasmic, enters first in the membrane. In contact mode the molecular structure of the periplasmic side of the transmembrane pigment-protein complex was observed. Using Föster's theory for describing the distance dependent energy transfer, we estimate the dipole strength for energy transfer between two neighboring LH2s, based on the architecture of the imaged unit cell.

Atomic force microscopy of the bacterial photosynthetic apparatus: plain pictures of an elaborate machinery

“…Interestingly, in reconstituted membranes of less extensively purified LH2 a minor fraction of LH1 complexes was co-purified and found assembled with LH2, providing first images of different protein complexes in one membrane (Scheuring et al 2001). Subsequently, a series of AFM studies, of purified and reconstituted proteins, was conducted on: LH2 (Stamouli et al 2003;Gonçalves et al 2005;Scheuring et al 2003a), LH1 (Bahatyrova et al 2004b), and LH1-RC core complexes Scheuring et al 2004aScheuring et al , 2005aSiebert et al 2004). Finally native membranes were imaged, first only containing LH1-RC core complexes (Scheuring et al 2003a), then containing LH2 and LH1-RC core complexes (Scheuring et al 2004c(Scheuring et al , 2005bScheuring and Sturgis 2005;Gonçalves et al 2005;Bahatyrova et al 2004a).…”

Flexibility and Size Heterogeneity of the LH1 Light Harvesting Complex Revealed by Atomic Force Microscopy

“…The use of the atomic force microscope (AFM) to image the surface of two-dimensional crystals presents us with the opportunity to obtain high signal-to-noise data without the need for processing the data, and particularly without the need to obtain large, highly ordered crystals. Previous studies have amply illustrated the usefulness of AFM for imaging two-dimensional crystals of the LH2 complexes of Rubrivivax gelatinosus (16), R. sphaeroides (17), and R. acidophila (18). Scheuring et al (19) achieved the imaging of native membranes of Blastochloris viridis containing RC-LH1 complexes by AFM and were able to show that LH1 formed an ellipse round the RC, but that it became circular upon removal of the RC.…”

Preparation of reconstituted acetylcholine receptor membranes suitable for AFM imaging of lipid–protein interactions

“…Ordered two-dimensional arrays of membrane proteins including rhodopsin, porins, bacterial hexagonally packed intermediate layers, photosynthetic core-complexes and connexons are particularly amenable to high resolution AFM imaging (Arechaga and Fotiadis, 2007;Buzhynskyy et al, 2007;Fotiadis et al, 2003;Muller et al, 1997;Scheuring, 2006;Stamouli et al, 2003;Thimm et al, 2005). These studies have provided unprecedented structural data for supramolecular assemblies of membrane proteins in aqueous buffer under close to physiolog-ical conditions, where conformational changes and the effects of factors such as pH and ligand binding can be readily studied.…”