Abstract
The mapping of the photosynthetic membrane of Rhodobacter sphaeroides by atomic force microscopy (AFM) revealed a unique organization of arrays of dimeric reaction center-light harvesting I-PufX (RC-LH1-PufX) core complexes surrounded and interconnected by light-harvesting LH2 complexes (Bahatyrova, S., Frese, R. N., Siebert, C. A., Olsen, J. D., van der Werf, K. O., van Grondelle, R., Niederman, R. A., Bullough, P. A., Otto, C., and Hunter, C. N. (2004) Nature 430, 1058-1062). However, membrane regions consisting solely of LH2 complexes were under-represented in these images because these small, highly curved areas of membrane rendered them difficult to image even using gentle tapping mode AFM and impossible with contact mode AFM. We report AFM imaging of membranes prepared from a mutant of R. sphaeroides, DPF2G, that synthesizes only the LH2 complexes, which assembles spherical intracytoplasmic membrane vesicles of approximately 53 nm diameter in vivo. By opening these vesicles and adsorbing them onto mica to form small, < or =120 nm, largely flat sheets we have been able to visualize the organization of these LH2-only membranes for the first time. The transition from highly curved vesicle to the planar sheet is accompanied by a change in the packing of the LH2 complexes such that approximately half of the complexes are raised off the mica surface by approximately 1 nm relative to the rest. This vertical displacement produces a very regular corrugated appearance of the planar membrane sheets. Analysis of the topographs was used to measure the distances and angles between the complexes. These data are used to model the organization of LH2 complexes in the original, curved membrane. The implications of this architecture for the light harvesting function and diffusion of quinones in native membranes of R. sphaeroides are discussed.
Highlights
Wide variety of integral membrane proteins that perform transport, sensing, motility, biosynthesis, energy generation, as well as energy harvesting in the case of phototrophic organisms
We have demonstrated that the comparatively disordered intracytoplasmic membrane from the model photosynthetic bacterium Rhodobacter sphaeroides can be successfully imaged by atomic force microscopy (AFM) [9] to reveal the hidden architecture that nature has developed to harvest, transfer, and utilize light energy with great efficiency
General Vesicle Morphology—Previous work to obtain high resolution AFM images of native membranes of R. sphaeroides used the mild detergent -DDM; for the membranes of the deletion strain DPF2G, we attempted to record data on nondetergent-treated membranes as well as those prepared by existing methodologies
Summary
The R. sphaeroides deletion strain DPF2G [16] was grown and the membranes were prepared according to the methods in Olsen et al [26] and further treated with the detergent -DDM (Glycon Biochemistry, GmbH Biotechnology, Germany), at 0.005 and 0.01% (w/v) concentration, following the protocol of Bahatyrova et al [9]. The sample was washed twice with 20 mM HEPES, pH 7.5, 100 mM KCl recording buffer. Olympus TR800PSA SiN cantilevers (Atomic Force GmbH, Mannheim, Germany), spring constant 0.15 N/m were used in a standard tapping mode liquid cell at operating frequencies of ϳ8.5 KHz, using a Nanoscope IV AFM and E scanner (VEECO). The images were recorded at scan frequencies of 0.5–1 Hz. The images were processed using the software supplied with the microscope (VEECO) and SPIP (Image Metrology). Angles, and areas were measured using ImageJ (open source software). The model vesicle was prepared using Chimera [27] (open source software)
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