Nanodissection and high-resolution imaging of the Rhodopseudomonas viridis photosynthetic core complex in native membranes by AFM

Abstract

In photosynthesis, highly organized multiprotein assemblies convert sunlight into biochemical energy with high efficiency. A challenge in structural biology is to analyze such supramolecular complexes in native membranes. Atomic force microscopy (AFM) with high lateral resolution, high signal-to-noise ratio, and the possibility to nanodissect biological samples is a unique tool to investigate multiprotein complexes at molecular resolution in situ.Here we present high-resolution AFM of the photosynthetic core complex in native Rhodopseudomonas viridis membranes. Topographs at 10-Å lateral and Ϸ1-Å vertical resolution reveal a single reaction center (RC) surrounded by a closed ellipsoid of 16 lightharvesting (LH1) subunits. Nanodissection of the tetraheme cytochrome (4Hcyt) subunit from the RC allows demonstration that the L and M subunits exhibit an asymmetric topography intimately associated to the LH1 subunits located at the short ellipsis axis. This architecture implies a distance distribution between the antenna and the RC compared with a centered location of the RC within a circular LH1, which may influence the energy transfer within the core complex. The LH1 subunits rearrange into a circle after removal of the RC from the core complex. P hotosynthetic organisms fuel their metabolism with light energy and have developed for this purpose an efficient apparatus for harvesting and converting sunlight into biochemical energy. The initial steps of photosynthesis, universal in photosynthetic bacteria, algae, and higher plants, comprise light absorption by a set of light-harvesting (LH) pigment-protein complexes and subsequent transfer of the excitation energy to the reaction center (RC), where charge separation across the membrane takes place. In photosynthetic bacteria, the so-called core complex, constituted of a RC intimately associated with LH1, performs these initial steps of the photosynthesis: light trapping and charge separation. The efficiency of the process demands high structural organization of the components. The spatial organization between LH1 and RC is still a matter of debate (1-8), and no information on their assembly in a native system is available. Complementary to structure determination of individual components at near-atomic resolution (9-11), one of the main challenges today is to describe the supramolecular organization of the photosynthetic machinery in native membranes (12). From a more general standpoint, structural biology is in need of a technique with a lateral resolution and signalto-noise ratio good enough to identify individual components of a multiprotein complex in situ (13).The atomic force microscope (14) has developed into a powerful tool in structural biology (15,16). It was demonstrated that topographs of two-dimensional crystals of membrane proteins can be acquired at subnanometer resolution (17). In addition, the high signal-to-noise ratio in raw data atomic force microscopy (AFM) topographs allows single-molecule imaging (16,18,19). Furthermore, by applying loading forc...

Structural Analysis of the Reaction Center Light-harvesting Complex I Photosynthetic Core Complex of Rhodospirillum rubrum Using Atomic Force Microscopy

“…Subsequently, a similar ellipticity was observed in AFM topographs of the membrane-bound Rps. viridis RC-LH1 complex, only to alter to a circular shape upon removal of the RC (16). Our topographs of the R. rubrum complex differ, in that ellipses are observed whether or not the RC is present.…”

“…2f, but qualitatively speaking, it means that the LH1 complex forms a slightly raised rim around the RC. Our measurements can be compared with those of Scheuring et al (16), who examined the periplasmic side of membranes from Rps. viridis, and found that the LH1 complex protruded from the membrane by ϳ10 Å.…”

“…However, this does not imply that there is a "correct" answer to the question of the shape of the LH1 complex, only that this shape depends on local forces exerted by neighboring complexes, which were clearly different in the orthorhombic and tetragonal two-dimensional crystals in Jamieson et al (6) and which are probably different again in Rps. viridis membranes (16). In fact, the shape of the LH1 complex is subject to subtle distortions; this fact is revealed because AFM provides information on individual molecules, whereas EM, for example, averages out such differences.…”

“…The aim of this work is to advance our knowledge of the photosynthetic core complex by building on the work of Scheuring et al (16) on the periplasmically exposed surface of the complex and by obtaining topographs of both the cytoplasmic (RC-H subunit) and periplasmic (c-type cytochrome) faces. This establishes the height of the RC-H subunit and the functionally crucial special pair bacteriochlorophylls in relation to the membrane bilayer.…”

“…Recently, the value of this technique has been demonstrated for the light-harvesting LH2 complexes of Rubrivivax gelatinosus and Rb. sphaeroides (14,15) and on membranes of Rhodopseudomonas viridis containing RC-LH1 complexes (16).…”