Molecular Mechanisms of Membrane Channel and Transporter

Intermediate-valence Yb-based quasicrystals and approximants

We are searching for intermediate-valence (IV) quasicrystals where IV ions are quasi-periodically arranged. Our previous study revealed that an icosahedral Au-Al-Yb quasicrystal forms an IV system [1]. X-ray absorption spectroscopy near the Yb L_3 edge indicated that the Yb ions in the quasicrystal assume a mean valence of 2.61, between a divalent state (4f^14, J = 0) and a trivalent one (4f^13, J = 7/2). Additionally we found that non-Fermi-liquid behaviour appears at low temperatures without doping, pressure, or field tuning. In this study we examined Au-M-Yb system, where M = Sn, Ge, and Ga. X-ray absorption spectroscopy measurements at SPring-8 (BL22XU) showed that each compounds forms IV system. The Yb valence values are respectively 2.27 for a Au-Sn-Yb 2/1 crystalline approximant, 2.24 for a Au-Ge-Yb 1/1 approximant, and 2.36 for a Au-Ga-Yb 1/1 approximant. By following the trend of Yb-based IV compounds, these valence values close to divalent suggest that the Yb 4f character in these compounds would be itinerant rather than localized one.

Resonant X-ray scattering in low quartz to characterize the polarization light

The variation with energy of the diffracted peak intensities around the absorption edges has been known for very long time. It is only very recently that resonant elastic x-ray scattering (REXS) experiments were performed on enantiomers [1], showing the sensitivity of this technique to study tiny features in these materials. In right and left low quartz, azimuthal scans of the (001) reflection intensity show the angular anisotropy by presenting a 3 fold periodicity. These scans are shifted and their amplitude oscillations vary when changing from right to left the enantiomer or when changing the light helicity. Our purpose is to show that azimuthal scans recording the (001) reflection intensity in right and left low quartz can be completely explained by the proper taking into account of the polarization characteristics of the incoming electromagnetic wave. More importantly, we show that such experiments are an excellent way to fully determine the light properties, when this one is not perfectly known. From these scans, we get 3 equations giving their relative shift, the ratio between their amplitude oscillations and the polarization rate. Consequently, without need of simulations because these equations only depend on the symmetry and the geometry, we are able to get the three unknowns which are the Stokes parameter values. Such characterization does not depend on energy, or absorbing atom atomic number. This is thus feasible at other edges or with other compounds, such as GeO2, having the same symmetry. This opens the possibility of characterizing the light polarization on a wide energy range. This study is supported by ab initio simulations on REXS and linear dichroism to validate our demonstration and to eliminate the other possibilities such as higher contribution in term of transition channels (E1E2 or E2E2) or birefringence effects.

X-ray crystallographic analysis of a MATE multidrug transporter from V. cholerae

MATE (Multidrug And Toxic compound Extrusion) family transporters are highly conserved from Bacteria to Eukarya including human, and export a broad range of xenobiotics using either a proton or a sodium ion gradient across the membrane. Especially in bacterial pathogens, MATE transporters contribute to their multiple drug resistance (MDR). To understand how MATE transporters export various substrates such as drugs and thus how pathogens acquire MDR, structural analyses are essential. The crystal structures of several MATE transporters from pathogens have been reported. However, because of the limited resolution and the lack of drug-MATE transporters complex structures, the recognition mechanism of various substrates and the coupling mechanism of the cation influx and the drug efflux have been poorly understood. Although the high-resolution structures of MATE transporters from non-pathogenic archaeal P. furiosus (PfMATE) have been reported, PfMATE shares low sequence identity with MATE transporters from pathogens such as V. cholerae. Therefore, further findings of the structural mechanism of MDR caused by MATE transporters from pathogens have been needed. To understand the substrate recognition and transport mechanism of MATE transporters from pathogens, we determined the crystal structures of one of MATE transporters from V. cholerae (VcMATE) at 2.5-2.7 Å resolutions using in meso crystallization method. The high-resolution structures of VcMATE show two distinct conformations, as observed in the structures of PfMATE, and reveal the large movement of transmembrane helix 1 and the putative substrate-binding site. The structures suggest that the bending of transmembrane helix 1 and the sequential collapse of the putative substrate-binding site induce the release of the bound substrate. This conformational change during the substrate transport may be a common mechanism among MATE transporters from pathogens to non-pathogens.

Increasing the probability of successful crystallization by using symmetric tag

Recent progress in the techniques of bio-macromolecular crystallography makes crystal structure analysis more powerful and useful for life science. The structure analysis of huge super-molecular (eukaryotic Ribosome, Vault etc.) and membrane proteins related to diseases were successful. Moreover, the structure/fragment drug design using crystal structure analysis method is also becoming reliable. However, crystallization still remains as a major bottleneck for determining bio-macromolecular structures, although many methods have been developed such as crystallization kits, crystallization robot, crystallizing in gel, space, and magnetic field, laser excitation, using antibody, modification of protein surface, and so forth. The current situation of crystallization is still dependent on the accidental method searching for a crystallization reagent and the growth environment since the methodology for obtaining a quality crystal for structure analysis is not established yet. Therefore, further development of more advanced crystallization methods is required to increase the probability of successful crystallization. In principal, probability of successful crystallization could be increased by polymerized molecules with 2 or 3-fold rotation symmetry [1]. We have solved more than 100 structures, and found some fragments which is isolated from core structure, and seem to contribute to form high quality crystal by forming a polymer with 2 or 3-fold axial symmetry. Thus, we developed a novel method by fussing target protein with crystallization tags named 2/3RS-tag. These 2/3RS-tags polymerize target proteins with 2 or 3-fold axial symmetry, and consequently accelerate formation of crystal. We will report and discuss this new method in this presentation.

Crystal structure of metallo-DNA duplex containing T-Hg(II)-T base pairs

The DNA duplex containing mercury-mediated base pairs (T-Hg(II)-T) is an attractive biomacromolecular nanomaterials. In a recent study, it was confirmed that the Hg(II) ion significantly stabilizes a DNA duplex by binding selectively to a T-T mispair [1]. Based on the phenomenon observed, a DNA-based sensing system that selectively and sensitively detects Hg(II) ions in aqueous solution was developed [2]. In the present study, we have solved the first crystal structure of a B-form DNA duplex containing two consecutive T-Hg(II)-T base pairs [3]. The Hg(II) ion occupies the center between two T residues. The geometry of the T-Hg(II)-T base pair is very similar to that of the canonical Watson-Crick base pairs. The distance of N3-Hg(II) bond is 2.0 Å, suggesting that the N3 nitrogen releases an imino-proton even at neutral pH (pKa of N3 position of T is 9.8) and directly bonds to Hg(II). In the B-form DNA, the helical axis runs through the center of base pairs, and the Hg(II) ions are therefore aligned along the helical axis. The distance between the two neighboring Hg(II) ions is 3.3 Å. The relatively short Hg(II)-Hg(II) distance indicates that the metallophilic attraction could exit between them and may stabilize the B-form duplex. To support this, the DNA duplex is largely distorted and adopts an unusual non-helical conformation in the absence of Hg(II). In conclusion, the Hg(II) ion is essential for maintaining the B-form conformation of the DNA duplex containing T-T mispairs. The structure of the Hg(II)-DNA hybrid duplex itself and the Hg(II)-induced structural switching from the non-helical form to the B-form provide the basis for the structure-based design of metal-conjugated nucleic acid nanomaterials.