Search

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 (1,2). Therefore, we usually supplement the information on hydrogen atoms and valence electrons in proteins with preexisting 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 (3). 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 [Fe.sub.4] [S.sub.4] 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., Fine structural information, including the positions of hydrogen atoms and distributions of valence electrons, is essential for understanding the full properties of protein molecules; in particular, of electron transfer metalloproteins [...]

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, toour knowledge, of a LH1-RCcomplex, namely that of a [Ca.sup.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.sup.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., The photosynthetic light reaction is a process by which light energy is converted into chemical energy. The primary process of the light reaction is the absorption of sunlight by a [...]

Tetrathionate hydrolase (4THase) plays an important role in dissimilatory sulfur oxidation in the acidophilic iron‐ and sulfur‐oxidizing bacterium Acidithiobacillus ferrooxidans. The structure of recombinant 4THase from A. ferrooxidans (Af‐Tth) was determined by X‐ray crystallography to a resolution of 1.95 Å. Af‐Tth is a homodimer, and its monomer structure exhibits an eight‐bladed β‐propeller motif. Two insertion loops participate in dimerization, and one loop forms a cavity with the β‐propeller region. We observed unexplained electron densities in this cavity of the substrate‐soaked structure. The anomalous difference map generated using diffraction data collected at a wavelength of 1.9 Å indicated the presence of polymerized sulfur atoms. Asp325, a highly conserved residue among 4THases, was located near the polymerized sulfur atoms. 4THase activity was completely abolished in the site‐specific Af‐Tth D325N variant, suggesting that Asp325 plays a crucial role in the first step of tetrathionate hydrolysis. Considering that the Af‐Tth reaction occurs only under acidic pH, Asp325 acts as an acid for the tetrathionate hydrolysis reaction. The polymerized sulfur atoms in the active site cavity may represent the intermediate product in the subsequent step. PDB Code(s): 6L8A and 7CQY; [ABSTRACT FROM AUTHOR]

The outer membrane lipoprotein NlpE functions in stress response by activating the Cpx signal transduction pathway. The nonlipidated Cys1Ala mutant of NlpE with a C-terminal His tag from Escherichia coli was constructed, overexpressed and purified. Crystals of NlpE were grown in two distinct forms by the sitting-drop vapour-diffusion method at 298 K. The tetragonal crystals diffracted to 2.8 Å resolution and belong to space group P43212. The monoclinic crystals diffracted to 3.0 Å resolution and belong to space group C2. Initial phases were obtained from a tetragonal crystal of selenomethionylated protein by the MAD method. [ABSTRACT FROM AUTHOR]

[NiFe]-hydrogenase from Desulfovibrio vulgaris Miyazaki F is an O 2 -sensitive enzyme that is inactivated in the presence of O 2 but the oxidized enzyme can recover its catalytic activity by reacting with H 2 under anaerobic conditions. Here, we report the first neutron structure of [NiFe]-hydrogenase in its oxidized state, determined at a resolution of 2.20 Å. This resolution allowed us to reinvestigate the structure of the oxidized active site and to observe the positions of protons in several short hydrogen bonds. X-ray anomalous scattering data revealed that a part of the Ni ion is dissociated from the active site Ni-Fe complex and forms a new square-planar Ni complex, accompanied by rearrangement of the coordinated thiolate ligands. One of the thiolate Sγ atoms is oxidized to a sulfenate anion but remains attached to the Ni ion, which was evaluated by quantum chemical calculations. These results suggest that the square-planar complex can be generated by the attack of reactive oxygen species derived from O 2 , as distinct from one-electron oxidation leading to a conventional oxidized form of the Ni-Fe complex. Another major finding of this neutron structure analysis is that the Cys17 S thiolate Sγ atom coordinating to the proximal Fe-S cluster forms an unusual hydrogen bond with the main-chain amide N atom of Gly19 S with a distance of 3.25 Å, where the amide proton appears to be delocalized between the donor and acceptor atoms. This observation provides insight into the contribution of the coordinated thiolate ligands to the redox reaction of the Fe-S cluster., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Tetrathionate hydrolase (4THase) plays an important role in dissimilatory sulfur oxidation in the acidophilic iron- and sulfur-oxidizing bacterium Acidithiobacillus ferrooxidans. The structure of recombinant 4THase from A. ferrooxidans (Af-Tth) was determined by X-ray crystallography to a resolution of 1.95 Å. Af-Tth is a homodimer, and its monomer structure exhibits an eight-bladed β-propeller motif. Two insertion loops participate in dimerization, and one loop forms a cavity with the β-propeller region. We observed unexplained electron densities in this cavity of the substrate-soaked structure. The anomalous difference map generated using diffraction data collected at a wavelength of 1.9 Å indicated the presence of polymerized sulfur atoms. Asp325, a highly conserved residue among 4THases, was located near the polymerized sulfur atoms. 4THase activity was completely abolished in the site-specific Af-Tth D325N variant, suggesting that Asp325 plays a crucial role in the first step of tetrathionate hydrolysis. Considering that the Af-Tth reaction occurs only under acidic pH, Asp325 acts as an acid for the tetrathionate hydrolysis reaction. The polymerized sulfur atoms in the active site cavity may represent the intermediate product in the subsequent step., (© 2020 The Protein Society.)

MicroRNA (miRNA)-guided argonaute (Ago) controls gene expression upon binding to the 3' UTR of mRNA. The miRNA function can be competitively inhibited by single-stranded anti-miRNA oligonucleotides (AMOs). In this study, we constructed a novel type of AMO flanked by interstrand cross-linked 2'-O-methylated RNA duplexes (CLs) that confer a stable helical conformation. Compared with other structured AMOs, AMO flanked by CLs at the 5' and 3' termini exhibited much higher inhibitory activity in cells. Anti-miRNA activity, nuclease resistance, and miRNA modification pattern distinctly differed according to the CL-connected positions in AMOs. Moreover, we found that the 3'-side CL improves nuclease resistance, whereas the 5'-side CL contributes to stable binding with miRNA in Ago upon interaction with the 3' part of miRNA. These structure-function relationship analyses of AMOs provide important insights into the function control of Ago-miRNA complexes, which will be useful for basic miRNA research as well as for determining therapeutic applications of AMO., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)