Biomedical subjects

A Haouz

Publications and source records attributed to A Haouz.

10 recordsLinked to original sources

PubMed2006

Implementation of semi-automated cloning and prokaryotic expression screening: the impact of SPINE.

The implementation of high-throughput (HTP) cloning and expression screening in Escherichia coli by 14 laboratories in the Structural Proteomics In Europe (SPINE) consortium is described. Cloning efficiencies of greater than 80% have been achieved for the three non-ligation-based cloning techniques used, namely Gateway, ligation-indendent cloning of PCR products (LIC-PCR) and In-Fusion, with LIC-PCR emerging as the most cost-effective. On average, two constructs have been made for each of the approximately 1700 protein targets selected by SPINE for protein production. Overall, HTP expression screening in E. coli has yielded 32% soluble constructs, with at least one for 70% of the targets. In addition to the implementation of HTP cloning and expression screening, the development of two novel technologies is described, namely library-based screening for soluble constructs and parallel small-scale high-density fermentation.

Amino Acid Sequence↗

PubMed2006

The impact of protein characterization in structural proteomics.

Protein characterization plays a role in two key aspects of structural proteomics. The first is the quality assessment of the produced protein preparations. Obtaining well diffracting crystals is one of the major bottlenecks in the structure-determination pipeline. Often, this is caused by the poor quality of the protein preparation used for crystallization trials. Hence, it is essential to perform an extensive quality assessment of the protein preparations prior to crystallization and to use the results in the evaluation of the process. Here, a protein-production and crystallization strategy is proposed with threshold values for protein purity (95%) and monodispersity (85%) below which a further optimization of the protein-production process is strongly recommended. The second aspect is the determination of protein characteristics such as domains, oligomeric state, post-translational modifications and protein-protein and protein-ligand interactions. In this paper, applications and new developments of protein-characterization methods using MS, fluorescence spectroscopy, static light scattering, analytical ultracentrifugation and small-angle X-ray scattering within the EC Structural Proteomics in Europe contract are described. Examples of the application of the various methods are given.

Crystallization↗

PubMed2006

Application of the use of high-throughput technologies to the determination of protein structures of bacterial and viral pathogens.

The Structural Proteomics In Europe (SPINE) programme is aimed at the development and implementation of high-throughput technologies for the efficient structure determination of proteins of biomedical importance, such as those of bacterial and viral pathogens linked to human health. Despite the challenging nature of some of these targets, 175 novel pathogen protein structures (approximately 220 including complexes) have been determined to date. Here the impact of several technologies on the structural determination of proteins from human pathogens is illustrated with selected examples, including the parallel expression of multiple constructs, the use of standardized refolding protocols and optimized crystallization screens.

Animals↗

PubMed2003

Mycobacterium tuberculosis thymidylate kinase: structural studies of intermediates along the reaction pathway.

Mycobacterium tuberculosis TMP kinase (TMPK(Mtub)) represents a promising target for developing drugs against tuberculosis because the configuration of its active site is unique in the TMPK family. To help elucidate the phosphorylation mechanism employed by this enzyme, structural changes occurring upon binding of substrates and subsequent catalysis were investigated by protein crystallography. Six new structures of TMPK(Mtub) were solved at a resolution better than 2.3A, including the first structure of an apo-TMPK, obtained by triggering catalysis in a crystal of a TMPK(Mtub)-TMP complex, which resulted in the release of the TDP product. A series of snapshots along the reaction pathway is obtained, revealing the closure of the active site in going from an empty to a fully occupied state, suggestive of an induced-fit mechanism typical of NMPKs. However, in TMPK(Mtub) the LID closure couples to the binding with an unusual location for a magnesium ion coordinating TMP in the active site. Our data suggest strongly that this ion is required for catalysis, acting as a clamp, possibly in concert with Arg95, to neutralise electrostatic repulsion between the anionic substrates, optimise their proper alignment and activate them through direct and water-mediated interactions. The 3'-hydroxyl moiety of TMP, critical to metal stabilisation, appears to be a target of choice for the design of potent inhibitors. On the other hand, the usual NTP-bound magnesium is not seen in our structures and Arg14, a P-loop residue unique to TMPK(Mtub), may take over its role. Therefore, TMPK(Mtub) seems to have swapped the use of a metal ion as compared with e.g. human TMPK. Finally, TTP was observed in crystals of TMPK(Mtub), locked by Arg14, thus providing a structural explanation for the observed inhibitory effect of TTP putatively involved in a mechanism of feedback regulation of the enzymatic activity.

Catalysis↗

PubMed2001

Involvement of protein dynamics in enzyme stability. The case of glucose oxidase.

Dynamics of glucose oxidase immobilized and in solution were compared through their tryptophan fluorescence spectra, decay times and quenching by acrylamide. Energy barrier for thermal inactivation and melting temperature of both soluble and immobilized enzyme were also measured. Data show that the fluctuation amplitude is at the origin of protein instability.

Enzyme Stability↗

PubMed2000

Resolved fluorescence of the two tryptophan residues in horse apomyoglobin.

The composite fluorescence emission from the two tryptophans (W7 and W14) of horse heart apomyoglobin was explored by fluorescence quenching experiments. The fluorescence of the W7 residue is the only one involved in the quenching by iodide or trichloroethanol (TCE) titration. The fluorescence contribution of W7 is 49% of the total apomyoglobin emission, and its spectrum is red-shifted compared to the W14 emission. The fluorescence decay of Trp residues gives an average fluorescence lifetime of 2.06 ns for W14 and 2.84 ns for W7. The static fluorescence quenching by TCE was used to monitor the individual motions of the two tryptophans in apomyoglobin. The short correlation time of W7 (rho = 3 ns) explains why this residue can experience various environments without having to assume the existence of several protein conformations occurring during its lifetime emission.

Animals↗

PubMed1999

Heterogeneous motions within human apohemoglobin.

Conservation of the secondary and tertiary protein organization of human apohemoglobin was observed at temperatures ranging from 7 to 25 degrees C using CD spectra in the far-UV (200-250 nm) and near-UV (250-300 nm) regions. The dynamics of apohemoglobin were probed using fluorescence quenching experiments on the Trp residues and an extrinsic dye (ANS or bis-ANS) located in the heme cavities. The long decay time of the dye emission (> 10 ns) reveals the dynamics of the protein matrix averaged over the whole molecule. The short decay time of the Trp residue emission (congruent with 3 ns) probes the dynamics of their close vicinities. When the temperature rises from 10 to 20 degrees C, the average intraproteic motions throughout the whole apohemoglobin matrix are greatly accelerated, whereas the hydrophobic protein regions around the alpha14, beta15 and beta37 Trp residues appear much less animated. These dynamic differences between the behavior of the softer matrix and the packed rigid regions containing the tryptophans could be one of the requisites for apohemoglobin stability. We suspect that the highly rigid tryptophan domains in human apohemoglobin are likely to be knots.

Adult↗

PubMed1998

Solvent effects on horse apomyoglobin dynamics.

The effects of the solvent conditions (buffer pH 9, 8, or 7 or buffer pH 6.5 alone or mixed with 3.2% ethanol or 6.2% formamide) on the protein dynamics of horse apomyoglobin were investigated through tryptophan fluorescence quenching, spectra, and decay properties. Raising the pH (which induces discontinuous protein conformation changes) increases the structural fluctuations inside the hydrophobic A, G, and H helix core. Mixed solutions containing either 3.2% ethanol or 6.2% formamide (which redistribute water molecules on the protein surface) produce protein dynamics changes in the vicinity of the two Trp residues, without inducing particular constraints on these very residues. Formamide increases, in the same way, the polarity and the protein flexibility while ethanol reduces both. The present fluorescence work also shows that, whatever the outside solvent, the two Trp residues W7 and W14, embedded in the A, G, and H helix core, are equally and statistically reached by small molecules diffusing inside the protein matrix. Hydrogen-tritium exchange measurements on the protein in mixed solvents reveal that the dynamics of the A, G, and H helix cluster and of the B and E helixes are greatly influenced by the nature of the outside medium. A small amount of formamide in the buffer increases the protein fluctuations while an ethanol-water mixture reduces them. We suggest that the hydratation state of the protein surface could be the relevant parameter of the protein dynamics.

Animals↗

PubMed1998

Dynamic and structural properties of glucose oxidase enzyme.

The catalytic oxidation of beta-D-glucose by the enzyme glucose oxidase involves a redox change of the flavin coenzyme. The structure and the dynamics of the two extreme glucose oxidase forms were studied by using infrared absorption spectroscopy of the amide I'band, tryptophan fluorescence quenching and hydrogen isotopic exchange. The conversion of FAD to FADH2 does not change the amount of alpha-helix present in the protein outer shell, but reorganizes a fraction of random coil to beta-sheet structure. The dynamics of the protein interior vary with the redox states of the flavin without affecting the motions of the structural elements near the protein surface. From the structure of glucose oxidase given by X-ray crystallography, these results suggest that the dynamics of the interface between the two monomers are involved in the catalytic mechanism.

Aspergillus niger↗

PubMed1994

Assay of dehydrogenases with an O2-consuming biosensor.

Coimmobilization of L-lactate oxidase (LOD) and L-lactate dehydrogenase (LDH) on a film bound to an oxygen electrode makes it possible, by recycling L-lactate and pyruvate, to lower their detection limit to 10 nM. With LOD immobilized alone and LDH in solution, easy LDH measurements were performed. The detection limit was 1 IUl-1. Similarly, this mixed biosensor, using an LDH sensitive to mercurials, was used for p-chloromercuribenzoate (PCMB) detection. Coimmobilization of LOD + LDH with an NAD(+)-dependent dehydrogenase allows the measurements of all dehydrogenases. NAD+ is recycled. The detection limit was 50 nM. Applications can be found in medicine, the food industry, and the environment.

Animals↗