Site-specific relaxation of peptide bond planarity induced by electrically attracted proton/deuteron observed by neutron crystallography.

In structural biology, peptide bonds, fundamental linkages between hundreds of amino acids, of which a protein molecule is composed, have been commonly treated as a plane structure just as Linus Pauling et al. proposed. In this paper, a site-specific peptide bond relaxation mechanism by deuterons whose localization has been suggested by neutron crystallography is proposed.

Current status and near future plan of neutron protein crystallography at J-PARC.

The IBARAKI Biological Crystal Diffractometer (iBIX) has been available for use at MLF (Material and Life Science Facility) in J-PARC (Japan Proton Accelerator Research Complex) since 2008. The development in state-of-the-art detector systems could enable iBIX to become one of the highest-performance neutron single-crystal diffractometers in the world. Here, together with other various developments, such as data reduction software, crystal growth, and new techniques in measurement coupled analysis, we provided new hydrogen and water structural data of several proteins and macromolecules.

Status of the neutron time-of-flight single-crystal diffraction data-processing software STARGazer.

The STARGazer data-processing software is used for neutron time-of-flight (TOF) single-crystal diffraction data collected using the IBARAKI Biological Crystal Diffractometer (iBIX) at the Japan Proton Accelerator Research Complex (J-PARC). This software creates hkl intensity data from three-dimensional (x, y, TOF) diffraction data. STARGazer is composed of a data-processing component and a data-visualization component.

Cryoprotectant-free high-pressure cooling and dynamic nuclear polarization for more sensitive detection of hydrogen in neutron protein crystallography.

To improve the sensitivity of hydrogen detection using neutrons, a proton-polarization technique together with a high-pressure cooling method is necessary. The highest pressure (200 MPa) used in the experiment described here enabled relatively large protein crystals to be cooled without any cryoprotectants while retaining the protein structure, and it was confirmed that high-pressure-cooled crystals diffracted to nearly the same resolution as flash-cooled small crystals soaked with cryoprotectants. Dynamic nuclear polarization was used as a proton-polarization technique for protein crystals, and ∼300 mg polycrystalline protein doped with TEMPOL gave a maximum proton polarization of 22.

"Newton's cradle" proton relay with amide-imidic acid tautomerization in inverting cellulase visualized by neutron crystallography.

Hydrolysis of carbohydrates is a major bioreaction in nature, catalyzed by glycoside hydrolases (GHs). We used neutron diffraction and high-resolution x-ray diffraction analyses to investigate the hydrogen bond network in inverting cellulase PcCel45A, which is an endoglucanase belonging to subfamily C of GH family 45, isolated from the basidiomycete Phanerochaete chrysosporium. Examination of the enzyme and enzyme-ligand structures indicates a key role of multiple tautomerizations of asparagine residues and peptide bonds, which are finally connected to the other catalytic residue via typical side-chain hydrogen bonds, in forming the "Newton's cradle"-like proton relay pathway of the catalytic cycle.

Protonation State and Hydration of Bisphosphonate Bound to Farnesyl Pyrophosphate Synthase.

Farnesyl pyrophosphate synthase (FPPS) catalyzes the condensation of isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate to FPP and is known to be a molecular target of osteoporosis drugs, such as risedronate (RIS), which is a nitrogen-containing bisphosphonate. The protonation states and hydration structure of RIS bound to FPPS were determined by neutron protein crystallography, which allows direct visualization of hydrogens and deuteriums. The structure analysis revealed that the phosphate groups of RIS were fully deprotonated with the abnormally decreased pKa, and that the roles of E93 and D264 consisted of canceling the extra negative charges upon the binding of ligands.

Insights into the Proton Transfer Mechanism of a Bilin Reductase PcyA Following Neutron Crystallography.

Phycocyanobilin, a light-harvesting and photoreceptor pigment in higher plants, algae, and cyanobacteria, is synthesized from biliverdin IXα (BV) by phycocyanobilin:ferredoxin oxidoreductase (PcyA) via two steps of two-proton-coupled two-electron reduction. We determined the neutron structure of PcyA from cyanobacteria complexed with BV, revealing the exact location of the hydrogen atoms involved in catalysis. Notably, approximately half of the BV bound to PcyA was BVH(+), a state in which all four pyrrole nitrogen atoms were protonated.

Crystallization and preliminary neutron diffraction experiment of human farnesyl pyrophosphate synthase complexed with risedronate.

Nitrogen-containing bisphosphonates (N-BPs), such as risedronate and zoledronate, are currently used as a clinical drug for bone-resorption diseases and are potent inhibitors of farnesyl pyrophosphate synthase (FPPS). X-ray crystallographic analyses of FPPS with N-BPs have revealed that N-BPs bind to FPPS with three magnesium ions and several water molecules. To understand the structural characteristics of N-BPs bound to FPPS, including H atoms and hydration by water, neutron diffraction studies were initiated using BIODIFF at the Heinz Maier-Leibnitz Zentrum (MLZ).

Physical properties, structure, and shape of radioactive Cs from the Fukushima Daiichi Nuclear Power Plant accident derived from soil, bamboo and shiitake mushroom measurements.

We conducted an elution experiment with contaminated soils using various aqueous reagent solutions and autoradiography measurements of contaminated bamboo shoots and shiitake mushrooms to determine the physical and chemical characteristics of radioactive Cs from the Fukushima Daiichi Nuclear Power Plant accident. Based on our study results and data in the literature, we conclude that the active Cs emitted by the accident fell to the ground as granular non-ionic materials. Therefore, they were not adsorbed or trapped by minerals in the soil, but instead physically adhere to the rough surfaces of the soil mineral particles.

Evaluation of performance for IBARAKI biological crystal diffractometer iBIX with new detectors.

The IBARAKI biological crystal diffractometer, iBIX, is a high-performance time-of-flight neutron single-crystal diffractometer for elucidating mainly the hydrogen, protonation and hydration structures of biological macromolecules in various life processes. Since the end of 2008, iBIX has been available to users' experiments supported by Ibaraki University. Since August 2012, an upgrade of the 14 existing detectors has begun and 16 new detectors have been installed for iBIX.

Fundamental studies for the proton polarization technique in neutron protein crystallography.

The isotope effect in conventional neutron protein crystallography (NPC) can be eliminated by the proton polarization technique (ppt). Furthermore, the ppt can improve detection sensitivity of hydrogen (relative neutron scattering length of hydrogen) by approximately eight times in comparison with conventional NPC. Several technical difficulties, however, should be overcome in order to perform the ppt.

Phase-diagram-guided method for growth of a large crystal of glycoside hydrolase family 45 inverting cellulase suitable for neutron structural analysis.

Neutron protein crystallography (NPC) is a powerful tool for determining the hydrogen position and water orientation in proteins, but a much larger protein crystal is needed for NPC than for X-ray crystallography, and thus crystal preparation is a bottleneck. To obtain large protein crystals, it is necessary to know the properties of the target protein in the crystallization solution. Here, a crystal preparation method of fungal cellulase PcCel45A is reported, guided by the phase diagram.

Hydrogen-bond network and pH sensitivity in human transthyretin.

Transthyretin (TTR) is a tetrameric protein. TTR misfolding and aggregation are associated with human amyloid diseases. Dissociation of the TTR tetramer is believed to be the rate-limiting step in the amyloid fibril formation cascade.

Neutron and X-ray crystallographic analysis of the human α-thrombin-bivalirudin complex at pD 5.0: protonation states and hydration structure of the enzyme-product complex.

The protonation states and hydration structures of the α-thrombin-bivalirudin complex were studied by joint XN refinement of the single crystal X-ray and neutron diffraction data at resolutions of 1.6 and 2.8Å, respectively.

Quaternary structure, aggregation and cytotoxicity of transthyretin.

Transthyretin (TTR) with a Ser112-to-Ile mutation is known to cause amyloidosis with severe cardiomyopathy. We investigated the quaternary structure, aggregation and cytotoxicity of the S112I variant. This variant exists as a dimer at physiological pH, self-assembles into spherical aggregates and induces cell death in human neuroblastoma IMR-32 cells.

Hydrogen-bond network and pH sensitivity in transthyretin: Neutron crystal structure of human transthyretin.

Transthyretin (TTR) is a tetrameric protein associated with human amyloidosis. In vitro, the formation of amyloid fibrils by TTR is known to be promoted by low pH. Here we show the neutron structure of TTR, focusing on the hydrogen bonds, protonation states and pH sensitivities.

X-ray and neutron protein crystallographic analysis of the trypsin-BPTI complex.

In this work, the crystal structure of the β-trypsin-bovine pancreatic trypsin inhibitor (BPTI) complex was refined and the D and H atoms in the complex were identified using data from both 1.6 Å resolution X-ray diffraction and 2.15 Å resolution neutron diffraction.

Neutron structure analysis using the IBARAKI biological crystal diffractometer (iBIX) at J-PARC.

The IBARAKI Biological Crystal Diffractometer (iBIX), a new diffractometer for protein crystallography at the next-generation neutron source at J-PARC (Japan Proton Accelerator Research Complex), has been constructed and has been operational since December 2008. Preliminary structure analyses of organic crystals showed that iBIX has high performance even at 120 kW operation and the first full data set is being collected from a protein crystal.

[Beginning of use of a new biological neutron diffractometer (iBIX) in J-PARC].

Ibaraki Prefectural Government together with Ibaraki University and Japan Atomic Energy Agency (JAEA) has almost finished constructing a time-of-flight (TOF) neutron diffractometer for biological macromolecules for industrial use at J-PARC, IBARAKI Biological Crystal Diffractometer (iBIX). Since 2009, Ibaraki University has been asked to operate this machine in order for users to do experiments by Ibaraki Prefecture. The diffractometer is designed to cover sample crystals which have their cell edges up to around 150 A.

A neutron crystallographic analysis of T6 porcine insulin at 2.1 A resolution.

Neutron diffraction data for T(6) porcine insulin were collected to 2.1 A resolution from a single crystal partly deuterated by exchange of mother liquor. A maximum-likelihood structure refinement was undertaken using the neutron data and the structure was refined to a residual of 0.

Low-barrier hydrogen bond in photoactive yellow protein.

Low-barrier hydrogen bonds (LBHBs) have been proposed to play roles in protein functions, including enzymatic catalysis and proton transfer. Transient formation of LBHBs is expected to stabilize specific reaction intermediates. However, based on experimental results and theoretical considerations, arguments against the importance of LBHB in proteins have been raised.

To flip or not to flip? Assessing the inversion barrier of the tetraphenylene framework with enantiopure 2,15-dideuteriotetraphenylene and 2,7-dimethyltetraphenylene.

Two chiral tetraphenylenes, 2,15-dideuteriotetraphenylene (7) and 2,7-dimethyltetraphenylene (15) were synthesized and resolved to address the tetraphenylene inversion barrier problem. Neutron diffraction investigation of enantiopure 7 showed that the molecule retained its chirality integrity during its synthesis from enantiopure precursors and therefore rules out the possibility of the tetraphenylene framework possessing a low-energy barrier to inversion. Thermal study on 15 and tetraphenylene 1 further revealed that their inversion barriers were not overcome up to 600 degrees C, at which temperature these compounds underwent skeletal contraction into triphenylene with activation energies of 62.

An abnormal pK(a) value of internal histidine of the insulin molecule revealed by neutron crystallographic analysis.

Insulin is stored in pancreatic beta-cell as hexameric form with Zn2+ ions, while the hormonally active form is monomer. The hexamer requires the coordination of Zn2+ ions to the HisB10. In order to reveal the mechanism of the hexamerization of insulin, we investigated the Zn2+ free insulin at pD6.