The light-harvesting core antenna (LH1) and the reaction centre (RC) of purple photosynthetic bacteria form a supramolecular complex (LH1-RC) to use sunlight energy in a highly efficient manner. Here we report the first near-atomic structure, to our knowledge, of a LH1-RC complex, namely that of a Ca(2+)-bound complex from Thermochromatium tepidum, which reveals detailed information on the arrangement and interactions of the protein subunits and the cofactors. The RC is surrounded by 16 heterodimers of the LH1 αβ-subunit that form a completely closed structure. The Ca(2+) ions are located at the periplasmic side of LH1. Thirty-two bacteriochlorophyll and 16 spirilloxanthin molecules in the LH1 ring form an elliptical assembly. The geometries of the pigment assembly involved in the absorption characteristics of the bacteriochlorophyll in LH1 and excitation energy transfer among the pigments are reported. In addition, possible ubiquinone channels in the closed LH1 complex are proposed based on the atomic structure.

Thermophilic purple sulfur bacterium, Thermochromatium tepidum, can grow at temperatures up to 58 degrees C and exhibits an unusual Qy absorption at 915 nm for the core light-harvesting complex (LH1), an approximately 35-nm red shift from those of its mesophilic counterparts. We demonstrate in this study, using a highly purified LH1-reaction center complex, that the LH1 Qy transition is strongly dependent on metal cations and Ca2+ is involved in the unusual red shift. Removal of the Ca2+ resulted in formation of a species with the LH1 Qy absorption at 880 nm, and addition of the Ca2+ to the 880-nm species recovered the native 915-nm form. Interchange between the two forms is fully reversible. Based on spectroscopic and isothermal titration calorimetry analyses, the Ca2+ binding to the LH1 complex was estimated to occur in a stoichiometric ratio of Ca2+/alphabeta-subunit = 1:1 and the binding constant was in 10(5) m(-1) order of magnitude, which is comparable with those for EF-hand Ca2+-binding proteins. Despite the high affinity, conformational changes in the LH1 complex upon Ca2+ binding were small and occurred slowly, with a typical time constant of approximately 6 min. Replacement of the Ca2+ with other metal cations caused blue shifts of the Qy bands depending on the property of the cations, indicating that the binding site is highly selective. Based on the amino acid sequences of the LH1 complex, possible Ca2+-binding sites are proposed that consist of several acidic amino acid residues near the membrane interfaces of the C-terminal region of the alpha-polypeptide and the N-terminal region of the beta-polypeptide.

The results of stabilizing neoclassical tearing modes (NTMs) with electron cyclotron current drive (ECCD) in JT-60U are described with the emphasis on the effectiveness of the stabilization. The range of the minimum EC wave power needed for complete stabilization of an m/n = 2/1 NTM was experimentally identified for two regimes using unmodulated ECCD to clarify the NTM behaviors with different plasma parameters: 0.2 < j EC /j BS < 0.4 for W sat /d EC ∼ 3 and W sat /W marg ∼ 2, and 0.35 < j EC /j BS < 0.46 for W sat /d EC ∼ 1.5 and W sat /W marg ∼ 2. Here, m and n are the poloidal and toroidal mode numbers; j EC and j BS the EC-driven current density and bootstrap current density at the mode rational surface; W sat , W marg and d EC the full island width at saturation, marginal island width and full width at the half maximum of the ECCD deposition profile, respectively. Stabilization of a 2/1 NTM using modulated ECCD synchronized with a mode rotation of about 5 kHz was performed, in which it was found that the stabilization effect degrades when the phase of the modulation deviates from that of the ECCD at the island O-point. The decay time of magnetic perturbation amplitude due to the ECCD increases by 50% with a phase shift of ±50 • from the O-point ECCD, thus revealing the importance of the phasing of modulated ECCD. For near X-point ECCD, the NTM amplitude increases, revealing a destabilization effect. It was also found that modulated ECCD at the island O-point has a stronger stabilization effect than unmodulated ECCD by a factor of more than 2.

The fine structures of proteins, such as the positions of hydrogen atoms, distributions of valence electrons and orientations of bound waters, are critical factors for determining the dynamic and chemical properties of proteins. Such information cannot be obtained by conventional protein X-ray analyses at 3.0-1.5 Å resolution, in which amino acids are fitted into atomically unresolved electron-density maps and refinement calculations are performed under strong restraints. Therefore, we usually supplement the information on hydrogen atoms and valence electrons in proteins with pre-existing common knowledge obtained by chemistry in small molecules. However, even now, computational calculation of such information with quantum chemistry also tends to be difficult, especially for polynuclear metalloproteins. Here we report a charge-density analysis of the high-potential iron-sulfur protein from the thermophilic purple bacterium Thermochromatium tepidum using X-ray data at an ultra-high resolution of 0.48 Å. Residual electron densities in the conventional refinement are assigned as valence electrons in the multipolar refinement. Iron 3d and sulfur 3p electron densities of the Fe4S4 cluster are visualized around the atoms. Such information provides the most detailed view of the valence electrons of the metal complex in the protein. The asymmetry of the iron-sulfur cluster and the protein environment suggests the structural basis of charge storing on electron transfer. Our charge-density analysis reveals many fine features around the metal complex for the first time, and will enable further theoretical and experimental studies of metalloproteins.

SummaryThe coiled-coil motif mediates subunit oligomerization and scaffolding and underlies several fundamental biologic processes. Prohibitins (PHBs), mitochondrial inner membrane proteins involved in mitochondrial homeostasis and signal transduction, are predicted to have a coiled-coil motif, but their structural features are poorly understood. Here we solved the crystal structure of the heptad repeat (HR) region of PHB2 at 1.7-Å resolution, showing that it assembles into a dimeric, antiparallel coiled-coil with a unique negatively charged area essential for the PHB interactome in mitochondria. Disruption of the HR coiled-coil abolishes well-ordered PHB complexes and the mitochondrial tubular networks accompanying PHB-dependent signaling. Using a proximity-dependent biotin identification (BioID) technique in live cells, we mapped a number of mitochondrial intermembrane space proteins whose association with PHB2 relies on the HR coiled-coil region. Elucidation of the PHB complex structure in mitochondria provides insight into essential PHB interactomes required for mitochondrial dynamics as well as signal transduction.

Thermochromatium tepidum is a thermophilic purple sulfur photosynthetic bacterium collected from the Mammoth Hot Springs, Yellowstone National Park. A previous study showed that the light-harvesting-reaction center core complex (LH1-RC) purified from this bacterium is highly stable at room temperature (Suzuki, H., Hirano, Y., Kimura, Y., Takaichi, S., Kobayashi, M., Miki, K., and Wang, Z.-Y. (2007) Biochim. Biophys. Acta 1767, 1057-1063). In this work, we demonstrate that thermal stability of the Tch. tepidum LH1-RC is much higher than that of its mesophilic counterparts, and the enhanced thermal stability requires Ca 2؉ as a cofactor. Removal of the Ca 2؉ from Tch. tepidum LH1-RC resulted in a complex with the same degree of thermal stability as that of the LH1-RCs purified from mesophilic bacteria. The enhanced thermal stability can be restored by addition of Ca 2؉ to the Ca 2؉ -depleted LH1-RC, and this process is fully reversible. Interchange of the thermal stability between the two forms is accompanied by a shift of the LH1 Q y transition between 915 nm for the native and 880 nm for the Ca 2؉ -depleted LH1-RC. Differential scanning calorimetry measurements reveal that degradation temperature of the native LH1-RC is 15°C higher and the enthalpy change is about 28% larger than the Ca 2؉ -depleted LH1-RC. Substitution of the Ca 2؉ with other metal cations caused a decrease in thermal stability of an extent depending on the properties of the cations. These results indicate that Ca 2؉ ions play a dual role in stabilizing the structure of the pigment-membrane protein complex and in altering its spectroscopic properties, and hence provide insight into the adaptive strategy of this photosynthetic organism to survive in extreme environments using natural resources.Purple sulfur photosynthetic bacterium, Thermochromatium tepidum, was originally isolated from a hot springs in Yellowstone National Park and can grow anaerobically at optimum temperatures of 48 -50°C with an upper limit of 58°C (1). This is the highest temperature of all known purple bacteria (2). A number of soluble proteins purified from this organism have been shown to be thermostable with respect to their mesophilic counterparts. Ribulose-1,5-bisphosphate carboxylase/oxygenase, a key enzyme of the Calvin cycle, from Tch. tepidum was reported to be most catalytically active at 50°C, and it remained active at 60°C over 20 min, whereas the same enzyme from the closely related mesophilic bacterium Allochromatium vinosum completely lost its activity over the same period (3, 4). Similar behavior was observed for the high potential iron-sulfur proteins from the two bacteria (5, 6), and was attributed to subtle differences in the amino acid sequence and structure (7).The thermal stability mechanism of the membrane proteins is somewhat complicated because of complex protein-protein and protein-detergent/lipid interactions (8). In the case of reaction center (RC) 3 from Tch. tepidum, the thermal stability was shown to be strongly dependent on the type and compo...

A light-harvesting-reaction center (LH1-RC) core complex has been highly purified from a thermophilic purple sulfur bacterium, Thermochromatium tepidum. The bacteriochlorophyll (BChl) a molecules in the LH1 exhibit a Q(y) transition at 914 nm, more than 25 nm red-shift from those of its mesophilic counterparts. The LH1-RC complex was isolated in a monomeric form as confirmed by sucrose density gradient centrifugation, blue native PAGE and size-exclusion chromatography. Four subunits (L, M, H and a tetraheme cytochrome) in RC and two polypeptides (alpha and beta) in LH1 were identified. Spirilloxanthin was determined to be the predominant carotenoid in the core complex. The purified core complex was highly stable, no significant change in the LH1 Q(y) transition was observed over 10 days of incubation at room temperature in dark. Circular dichroism spectrum of the LH1 complex was characterized by low intensity and nonconservative spectral shape, implying a high symmetry of the large LH1 ring and interaction between the BChl a and carotenoid molecules. A dimeric feature of the BChl a molecules in LH1 was revealed by magnetic circular dichroism spectrum. Crystals of the core complex were obtained which diffracted X-rays to about 10 A.

NlpE, an outer membrane lipoprotein, functions during envelope stress responses in Gram-negative bacteria. In Escherichia coli, adhesion to abiotic surfaces has been reported to activate the Cpx pathway in an NlpE-dependent manner. External copper ions are also thought to activate the Cpx pathway mediated by NlpE. We determined the crystal structure of NlpE from E. coli at 2.6 A resolution. The structure showed that NlpE consists of two beta barrel domains. The N-terminal domain resembles the bacterial lipocalin Blc, and the C-terminal domain has an oligonucleotide/oligosaccharide-binding (OB) fold. From both biochemical analyses and the crystal structure, it can be deduced that the cysteine residues in the CXXC motif may be chemically active. Furthermore, two monomers in the asymmetric unit form an unusual 3D domain-swapped dimer. These findings indicate that tertiary and/or quaternary structural instability may be responsible for Cpx pathway activation.