Anne Théobald-Dietrich

The crystallization of biological macromolecules from precipitates: evidence for Ostwald ripening

Abstract Crystals were obtained by different methods under conditions where nucleation and growth occur from precipitated macromolecular material. The phenomenon was observed with compounds of different size and nature, such as thaumatin, concanavalin A, an α-amylase, a thermostable aspartyl-tRNA synthetase, the nucleo-protein complex between a tRNA Asp transcript and its cognate yeast aspartyl-tRNA synthetase, and tomato bushy stunt virus. In each system, after a rather rapid precipitation step at high supersaturation lasting one to several days, a few microcrystals appear after prolonged equilibration at constant temperature. With α-amylase, the virus and the thermostable synthetase, crystallization is accompanied by appearance of depletion zones around the growing crystals and growth of the largest crystals at the expense of the smaller ones. These features are evidences for crystal growth by Ostwald ripening. In the case of thaumatin, concanavalin A and the nucleo-protein complex, crystallization occurs by a phase transition mechanism since it is never accompanied by the disappearance of the smallest crystals. A careful analysis with thermostable aspartyl-tRNA synthetase indicates that its crystallization at 4°C under high supersaturation starts by a phase transition mechanism with the formation of small crystals within an amorphous protein precipitate. Ostwald ripening follows over a period of up to three/four months with a growth rate of about 0.8 A/s that is 13 times slower than that of crystals growing at 20°C in the absence of precipitate without ripening. At the end of the ripening process at 4°C, only one unique synthetase crystal remains per microassay with dimensions as large as 1 mm.

Ribozyme processed tRNA transcripts with unfriendly internal promoter for T7 RNA polymerase: production and activity

A limitation for a universal use of T7 RNA polymerase for in vitro tRNA transcription lies in the nature of the often unfavorable 5′‐terminal sequence of the gene to be transcribed. To overcome this drawback, a hammerhead ribozyme sequence was introduced between a strong T7 RNA polymerase promoter and the tDNA sequence. Transcription of this construct gives rise to a ‘transzyme’ molecule, the autocatalytic activity of which liberates a 5′‐OH tRNA transcript starting with the proper nucleotide. The method was optimized for transcription of yeast tRNATyr, starting with 5′‐C1, and operates as well for yeast tRNAAsp with 5′‐U1. Although the tRNAs produced by the transzyme method are not phosphorylated, they are fully active in aminoacylation with k cat and K m parameters quasi identical to those of their phosphorylated counterparts.

Crystallogenesis of biological macromolecules: facts and perspectives

This paper gives an overview of the science of crystals of biological macromolecules. The historical background of the field is outlined and the main achievements and open problems are discussed from both biological and physical-chemical viewpoints. Selected results, including data from the authors, illustrate this overview. The perspectives of crystallogenesis for structural biology, but also more general trends, are presented.

Crystallogenesis studies in microgravity with the Advanced Protein Crystallization Facility on SpaceHab-01

Abstract The Advanced Protein Crystallization Facility (APCF), a new protein crystallization device developed by ESA for the IML-2 Mission in 1994, was tested in its maiden flight on STS-57 Mission in SpaceHab-01 with a physico-chemical experiment on lysozyme crystallization. In pre-flight ground experiments, prior to the Shuttle Mission, the protocol for lysozyme crystallization with NaCl was based on its solubility diagram at 18°C and pH 4.5. Crystallization was conducted under microgravity in 25 APCF reactors using vapor diffusion, dialysis, and free liquid interface diffusion, with control on earth in 25 identical reactors. Identical supersaturation values were tested by the three crystallization techniques. Values of supersaturation derived from ground experiments allowed for conditions that yielded crystals in microgravity. The average number and size of crystals from the flight experiment and the earth control showed no significant difference; however many crystals were not free floating and grew on the walls of some of the protein chambers. The dialysis technique proved to be suitable, since no additional nucleation was generated by the membrane. Protein concentration measurements indicated that 13 days after activation of the experiment as much as 70–90% of the protein in supersaturated state had already crystallized. Data indicated differences in the crystallization behavior depending upon the crystallization set-up. Images of the protein chamber of 6 reactors, recorded during the flight, allowed us to evaluate the early stage of crystallization, to verify that recovered crystals had actually grown under microgravity conditions, and showed motions of crystals during the Mission. Using synchrotron radiation, resolution and rocking curve measurements of ground and space lysozyme crystals grown in APCF reactors showed no significant differences, although the values are much better than previously recorded diffraction limits and mosaicity data obtained with tetragonal lysozyme crystals grown in other set-ups and under different conditions. All controls foreseen throughout the microgravity experiment proved to be essential for the interpretation of the flight data, as concerning the effect of microgravity.

Exploring the aminoacylation function of transfer RNA by macromolecular engineering approaches. Involvement of conformational features in the charging process of yeast tRNAAsp

This report presents the conceptual and methodological framework that presently underlies the experiments designed to decipher the structural features in tRNA important for its aminoacylation by aminoacyl-tRNA synthetases. It emphasizes the importance of conformational features in tRNA for an optimized aminoacylation. This is illustrated by selected examples on yeast tRNA(Asp). Using the phage T7 transcriptional system, a series of tRNA(Asp) variants were created in which conformational elements were modified. It is shown that aspartyl-tRNA synthetase tolerates conformational variability in tRNA(Asp) at the level of the D-loop and variable region, of the tertiary Levitt base-pair 15-48 which can be inverted and in the T-arm in which residue 49 can be excised. However, changing the anticodon region completely abolishes the aspartylation capacity of the variants. Transplanting the phenylalanine identity elements into a different tRNA(Asp) variant presenting conformational characteristics of tRNA(Phe) converts this molecule into a phenylalanine acceptor but is less efficient than wild-type tRNA(Phe). This engineered tRNA completely loses its aspartylation capacity, showing that some aspartic acid and phenylalanine identity determinants overlap. The fact that chimeric tRNA(Asp) molecules with altered anticodon regions lose their aspartylation capacity demonstrates that this region is part of the aspartic acid identity of tRNA(Asp).

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.

Effects of macromolecular impurities and of crystallization method on the quality of eubacterial aspartyl‐tRNA synthetase crystals

Although macromolecular purity is thought to be essential for the growth of flawless protein crystals, only a few studies have investigated how contaminants alter the crystallization process and crystal quality. Likewise, the outcome of a crystallization process may vary with the crystallization method. Here, it is reported how these two variables affect the crystallogenesis of aspartyl-tRNA synthetase from the eubacterium Thermus thermophilus. This homodimeric enzyme (Mr=130,000) possesses a multi-domain architecture and crystallizes either in a monoclinic or an orthorhombic habit. Minute amounts of protein impurities alter to a different extent the growth of each crystal form. The best synthetase crystals are only obtained when the crystallizing solution is either enclosed in capillaries or immobilized in agarose gel. In these two environments convection is reduced with regard to that existing in an unconstrained solution.

Crystallogenesis in tRNA aminoacylation systems: how packing accounts for crystallization drawbacks with yeast aspartyl-tRNA synthetase

Two active forms of homodimeric aspartyl-tRNA synthetase from Saccharomyces cerevisiae differing in length at their N-terminus crystallize in the same orthorhombic space group (P4 1 2 1 2) with identical cell parameters. Initial studies were hampered by the poor and anisotropic diffraction of the crystals of enzyme extracted from yeast cells. Isotropic diffraction at higher resolution was obtained when crystals were grown from an engineered protein deprived of its 70 N-terminal amino acids. The present work describes the packing contacts in crystals of the shortened protein whose structure was solved at 2.3 A resolution. Each subunit of the enzyme develops two lattice interactions covering a surface of 670 A 2 , about 7-fold smaller than that of the interface between monomers. The smallest lattice interaction, covering 150 A 2 , brings the anticodon binding domain adjacent to the N-terminus of one monomer in contact with a loop from the active-site domain of a neighboring monomer. Modeling of the extension in the solvent channels shows that the 150 A 2 intermolecular contact is perturbed in protein molecules possessing a floppy appendix while their second and larger 520 A 2 contact area is unaffected. Altogether the packing organization explains the poor diffraction properties of the native enzyme crystals and the enhanced diffraction of the crystals of shortened synthetase.