We have determined the atomic structure of the bacteriochlorophyll c (BChl c) assembly in a huge light-harvesting organelle, the chlorosome of green photosynthetic bacteria, by solid-state NMR. Previous electron microscopic and spectroscopic studies indicated that chlorosomes have a cylindrical architecture with a diameter of Ϸ10 nm consisting of layered BChl molecules. Assembly structures in huge noncrystalline chlorosomes have been proposed based mainly on structure-dependent chemical shifts and a few distances acquired by solid-state NMR, but those studies did not provide a definite structure. Our approach is based on 13 C dipolar spindiffusion solid-state NMR of uniformly 13 C-labeled chlorosomes under magic-angle spinning. Approximately 90 intermolecular COC distances were obtained by simultaneous assignment of distance correlations and structure optimization preceded by polarization-transfer matrix analysis. It was determined from the Ϸ90 intermolecular distances that BChl c molecules form piggybackdimer-based parallel layers. This finding rules out the well known monomer-based structures. A molecular model of the cylinder in the chlorosome was built by using this structure. It provided insights into the mechanisms of efficient light harvesting and excitation transfer to the reaction centers. This work constitutes an important advance in the structure determination of huge intact systems that cannot be crystallized.spin diffusion ͉ distance analysis ͉ photosynthesis ͉ antenna complex ͉ excitation transfer P hotosynthesis is the primary energy source for all living organisms. Chlorophyll-protein complexes capture light energy in most photosynthetic systems. Their structures are well known (1, 2). However, there are other light-harvesting devices called chlorosomes, which contain bacteriochlorophyll (BChl) assemblies. No protein is present in the BChl assemblies in sharp contrast to the light-harvesting chlorophyll-protein complexes mentioned above. Chlorosomes are found only in green sulfur bacteria and green filamentous bacteria. They catch weak light in an environment (3). A green sulfur bacterium species found in a deep-sea hydrothermal vent (4), for example, uses the dim light of geothermal radiation for photosynthesis. The atomic structure of chlorosomes has not been determined, which has impeded structure-based study of their functions. Because the design of chlorosomes is completely different from that of other light-harvesting devices, elucidation of their structure provides insight into the light-harvesting mechanism involved.Freeze-fracture electron microscopy revealed that chlorosomes were oblong bodies filled with several rod-shaped elements and were attached to the cytoplasmic side of cell membranes (5, 6). The rod elements of Chlorobium limicola, the target of this work, are composed of BChl c. Light energy captured by BChl c in the rod elements is transferred to the reaction centers in the cytoplasmic membrane through BChl a in baseplate proteins (7). The BChl c molecule has two stereoisomers...

Magic angle spinning (MAS) solid-state nuclear magnetic resonance (NMR) is a powerful method for structure determination of insoluble biomolecules. However, structure determination by MAS solid-state NMR remains challenging because it is difficult to obtain a sufficient amount of distance restraints owing to spectral complexity. Collection of distance restraints from paramagnetic relaxation enhancement (PRE) is a promising approach to alleviate this barrier. However, the precision of distance restraints provided by PRE is limited in solid-state NMR because of incomplete averaged interactions and intermolecular PREs. In this report, the backbone structure of the B1 domain of streptococcal protein G (GB1) has been successfully determined by combining the CS-Rosetta protocol and qualitative PRE restraints. The derived structure has a Cα RMSD of 1.49 Å relative to the Xray structure. It is noteworthy that our protocol can determine the correct structure from only three cysteine-EDTA-Mn mutants because this number of PRE sites is insufficient when using a conventional structure calculation method based on restrained molecular dynamics and simulated annealing. This study shows that qualitative PRE restraints can be employed effectively for protein structure determination from a limited conformational sampling space using a protein fragment library. KeywordsSolid-state NMR Magic-angle spinning Protein structure Paramagnetic relaxation enhancement CS-Rosetta Electronic supplementary materialThe online version of this article

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A mixture of bacteriochlorophyll (BChl) c isomers was extracted from the cells of Chlorobium limicola that were grown in the media of 13C-enriched and natural-abundance isotopic compositions. The magic-angle spinning 13C NMR proton-driven spin-diffusion spectra were recorded with mixing times of 50, 100, and 250 ms for two different kinds of in vitro aggregates, one consisting of pure [13C]BChl c and the other consisting of a 1:1 mixture of [13C]BChl c and [12C]BChl c; those peaks whose intensities were reduced to approximately 1/4 by this dilution were assigned to intermolecular 13C-13C dipolar correlation peaks. On the other hand, the nearest-neighbor intermolecular carbon-carbon close contacts with distances of 4-6 A were simulated, to predict observed correlation peaks, for six different models of BChl c assembly. They include weakly overlapped monomers forming structure 1 and structure 2, strongly overlapped dimers forming straight and inclined columns, and weakly overlapped dimers forming aligned and displaced layers. Comparison between the observed correlation peaks and the predicted carbon-carbon close contacts, for both the macrocycles and the side chains, led us to a conclusion that the weakly overlapped dimers forming displaced layers are most likely the assembly of the BChl c molecules in the aggregate.