Christophe Charron

Non‐discriminating and discriminating aspartyl‐tRNA synthetases differ in the anticodon‐binding domain

In most organisms, tRNA aminoacylation is ensured by 20 aminoacyl‐tRNA synthetases (aaRSs). In eubacteria, however, synthetases can be duplicated as in Thermus thermophilus, which contains two distinct AspRSs. While AspRS‐1 is specific, AspRS‐2 is non‐discriminating and aspartylates tRNAAsp and tRNAAsn. The structure at 2.3 Å resolution of AspRS‐2, the first of a non‐discriminating synthetase, was solved. It differs from that of AspRS‐1 but has resemblance to that of discriminating and archaeal AspRS from Pyrococcus kodakaraensis. The protein presents non‐conventional features in its OB‐fold anticodon‐binding domain, namely the absence of a helix inserted between two β‐strands of this fold and a peculiar L1 loop differing from the large loops known to interact with tRNAAsp identity determinant C36 in conventional AspRSs. In AspRS‐2, this loop is small and structurally homologous to that in AsnRSs, including conservation of a proline. In discriminating Pyrococcus AspRS, the L1 loop, although small, lacks this proline and is not superimposable with that of AspRS‐2 or AsnRS. Its particular status is demonstrated by a loop‐exchange experiment that renders the Pyrococcus AspRS non‐discriminating.

Proteomic and 3D structure analyses highlight the C/D box snoRNP assembly mechanism and its control

During small nucleolar ribonucleoprotein complex assembly, a pre-snoRNP complex consisting only of protein components forms first, followed by displacement of the ZNHIT3 subunit when C/D snoRNAs bind and dynamic loading and unloading of RuvBL AAA+ ATPases.

Crystallization in the presence of glycerol displaces water molecules in the structure of thaumatin.

The intensely sweet protein thaumatin has been crystallized at 293 K in the presence of sodium tartrate and 25%(v/v) glycerol for X-ray diffraction data collection at 100 K. A comparison of the three-dimensional structure model derived from a crystal grown in the presence of glycerol with that of a control deprived of this additive reveals only minor changes in the overall structure but a approximately 20% reduction in the number of water molecules. X-ray topography analyses show that the overall quality of the crystals prepared in the presence of this cryoprotectant is enhanced.

The `Octopus' plate for protein crystallization under an electric field

A piece of equipment, adapted to a vapour-diffusion plate, has been designed to apply an external electrical field during the crystallization of protein samples.

Structure of thaumatin in a hexagonal space group: comparison of packing contacts in four crystal lattices.

The intensely sweet protein thaumatin has been crystallized in a hexagonal lattice after a temperature shift from 293 to 277 K. The structure of the protein in the new crystal was solved at 1.6 A resolution. The protein fold is identical to that found in three other crystal forms grown in the presence of crystallizing agents of differing chemical natures. The proportions of lattice interactions involving hydrogen bonds, hydrophobic or ionic groups differ greatly from one form to another. Moreover, the distribution of acidic and basic residues taking part in contacts also varies. The hexagonal packing is characterized by the presence of channels parallel to the c axis that are so wide that protein molecules can diffuse through them.

Purification, crystallization and preliminary X-ray diffraction data of L7Ae sRNP core protein from Pyrococcus abyssii

The L7Ae sRNP core protein from Pyrococcus abyssii was crystallized using the sitting-drop vapour-diffusion method. Crystals were obtained in the presence of MgCl(2), PEG 2000 MME and acetate buffer at pH 4.0. A native data set has been collected at 2.9 A resolution using a rotating-anode generator at room temperature. Crystals belong to the orthorhombic space group P2(1)2(1)2, with unit-cell parameters a = 70.7, b = 112.9, c = 34.8 A. There are two monomers of MW 14 200 Da per asymmetric unit and the packing density V(M) is 2.45 A(3) Da(-1). A molecular-replacement analysis gave solutions for the rotation and translation functions.

Crystal quality and differential crystal-growth behaviour of three proteins crystallized in gel at high hydrostatic pressure

Pressure is a non-invasive physical parameter that can be used to control and influence protein crystallization. It is also found that protein crystals of superior quality can be produced in gel. Here, a novel crystallization strategy combining hydrostatic pressure and agarose gel is described. Comparative experiments were conducted on hen and turkey egg-white lysozymes and the plant protein thaumatin. Crystals could be produced under up to 75-100 MPa (lysozymes) and 250 MPa (thaumatin). Several pressure-dependent parameters were determined, which included solubility and supersaturation of the proteins, number, size and morphology of the crystals, and the crystallization volume. Exploration of three-dimensional phase diagrams in which pH and pressure varied identified growth conditions where crystals had largest size and best morphology. As a general trend, nucleation and crystal-growth kinetics are altered and nucleation is always enhanced under pressure. Further, solubility of the lysozymes increases with pressure while that of thaumatin decreases. Likewise, changes in crystallization volumes at high and atmospheric pressure are opposite, being positive for the lysozymes and negative for thaumatin. Crystal quality was estimated by analysis of Bragg reflection profiles and X-ray topographs. While the quality of lysozyme crystals deteriorates as pressure increases, that of thaumatin crystals improves, with more homogeneous crystal morphology suggesting that pressure selectively dissociates ill-formed nuclei. Analysis of the thaumatin structure reveals a less hydrated solvent shell around the protein when pressure increases, with approximately 20% less ordered water molecules in crystals grown at 150 MPa when compared with those grown at atmospheric pressure (0.1 MPa). Noticeably, the altered water distribution is seen in depressurized crystals, indicating that pressure triggers a stable structural alteration on the protein surface while its polypeptide backbone remains essentially unaltered.

Crystal contacts engineering of aspartyl-tRNA synthetase from Thermus thermophilus: effects on crystallizability

To understand how surface residues in a protein structure influence crystal growth, packing arrangement and crystal quality, crystal surfaces were modified and crystallizability of seven different mutants investigated. The model was aspartyl-tRNA synthetase-1 from Thermus thermophilus, a homodimer (M(r) 122000) with a subunit of 580 amino acids. Engineering concerned modification of amino acids involved in packing contacts in the orthorhombic lattice (P2(1)2121) of the synthetase. Comparison of the crystallization behaviour of the mutants indicates a correlation between disruption/addition of packing interactions and crystallizability of the mutants: disruption or modification of lattice contacts prevents crystallization or leads to crystals of poor quality. In contrast, addition of potential contacts leads to well-shaped crystals of same space group and cell parameters than wild-type crystals.

From conventional crystallization to better crystals from space: a review on pilot crystallogenesis studies with aspartyl-tRNA synthetases

Aspartyl-tRNA synthetases were the model proteins in pilot crystallogenesis experiments. They are homodimeric enzymes of Mr approximately 125 kDa that possess as substrates a transfer RNA, ATP and aspartate. They have been isolated from different sources and were crystallized either as free proteins or in association with their ligands. This review discusses their crystallisability with emphasis to crystal quality and structure determination. Crystallization in low diffusivity gelled media or in microgravity environments is highlighted. It has contributed to prepare high-resolution diffracting crystals with better internal order as reflected by their mosaicity. With AspRS from Thermus thermophilus, the better crystalline quality of the space-grown crystals within APCF is correlated with higher quality of the derived electron density maps. Usefulness for structural biology of targeted methods aimed to improve the intrinsic physical quality of protein crystals is highlighted.

Fluoride glass planar optical waveguides

Abstract Ionic exchange techniques have been applied to multicomponent indium-based fluoride glasses in order to develop a gradient index structure having the properties of a planar optical waveguide. Several molten salts have been tested with the aim of exchanging Na+ by Li+ or K+. When the melt is an Li+-rich organic salt and when the glass sample contains about 15% Na+, the formation of a gradient index profile is characterized by ‘m-lines’ experiments. Depending on time and temperature of immersion, one, two or three modes of propagation have been detected in this new generation of waveguides. They are potential candidates for active and passive integrated optics operating in the visible and mid-infrared.