Wheat-germ cell-free production of prion proteins for solid-state NMR structural studies.

The expression of soluble, functional protein on a preparative scale poses a central challenge for structural studies. Cell-free protein expression has become a valuable alternative to cell-based methods, and allows today the expression of milligram quantities of protein. Its use is particularly attractive for NMR studies as it allows a multitude of isotopic labeling schemes.

Efficient bioconversion of lactose in milk and whey: immobilization and biochemical characterization of a beta-galactosidase from the dairy Streptococcus thermophilus LMD9 strain.

The gene encoding beta-galactosidase from dairy Streptococcus thermophilus strain LMD9 was cloned, sequenced and expressed in Escherichia coli. The recombinant enzyme was purified and showed high specific activity of 464 U/mg. This protein displays a homotetrameric arrangement composed of four 118 kDa monomers.

Prions: En route from structural models to structures.

The prion hypothesis states that the prion and non-prion form of a protein differ only in their 3D conformation and that different strains of a prion differ by their 3D structure. Recent technical developments have enabled solid-state NMR to address the atomic-resolution structures of full-length prions, and a first comparative study of two of them, HET-s and Ure2p, in fibrillar form, has recently appeared as a pair of companion papers. Interestingly, the two structures are rather different: HET-s features an exceedingly well-ordered prion domain and a partially disordered globular domain.

Prion fibrils of Ure2p assembled under physiological conditions contain highly ordered, natively folded modules.

The difference between the prion and the non-prion form of a protein is given solely by its three-dimensional structure, according to the prion hypothesis. It has been shown that solid-state NMR can unravel the atomic-resolution three-dimensional structure of prion fragments but, in the case of Ure2p, no highly resolved spectra are obtained from the isolated prion domain. Here, we demonstrate that the spectra of full-length fibrils of Ure2p interestingly lead to highly resolved solid-state NMR spectra.

Structural determinants of product specificity of sucrose isomerases.

The healthy sweetener isomaltulose is industrially produced from the conversion of sucrose by the sucrose isomerase SmuA from Protaminobacter rubrum. Crystal structures of SmuA in native and deoxynojirimycin complexed forms completed with modeling studies unravel the characteristics of the isomaltulose synthases catalytic pocket and their substrate binding mode. Comparison with the trehalulose synthase MutB highlights the role of Arg(298) and Arg(306) active site residues and surface charges in controlling product specificity of sucrose isomerases (isomaltulose versus trehalulose).

Subcellular localizations of the hepatitis C virus alternate reading frame proteins.

Alternate reading frame proteins (ARFPs) resulting either from frameshifting, from transcriptional slippage or from internal initiation in the +1 open reading frame (ORF) of hepatitis C virus (HCV) core protein coding sequence have been described in vitro. As an approach to study the roles of these proteins, we investigate the subcellular localization of ARFPs fused with the green fluorescent protein (GFP) either at their N- or C-terminus. Most GFP fusion products have a diffuse localization, as revealed by confocal microscopy.

Methyl proton contacts obtained using heteronuclear through-bond transfers in solid-state NMR spectroscopy.

A two-dimensional proton-mediated carbon-carbon correlation experiment that relies on through-bond heteronuclear magnetization transfers is demonstrated in the context of solid-state NMR of proteins. This new experiment, dubbed J-CHHC by analogy to the previously developed dipolar CHHC techniques, is shown to provide selective and sensitive correlations in the methyl region of 2D spectra of crystalline organic compounds. The method is then demonstrated on a microcrystalline sample of the dimeric protein Crh (2 x 10.

Conformational change induced by ATP binding in the multidrug ATP-binding cassette transporter BmrA.

ATP-binding cassette (ABC) transporters are involved in the transport of a wide variety of substrates, and ATP-driven dimerization of their nucleotide binding domains (NBDs) has been suggested to be one of the most energetic steps of their catalytic cycle. Taking advantage of the propensity of BmrA, a bacterial multidrug resistance ABC transporter, to form stable, highly ordered ring-shaped structures [Chami et al. (2002) J.

High-resolution solid-state MAS NMR of proteins-Crh as an example.

Solid-state NMR spectroscopy provides unique possibilities for the structural investigation of insoluble molecules at the atomic level. Recent efforts aim at solving the complete structures of biological macromolecules using high-resolution magic angle spinning NMR. Structurally homogenous samples of [(13)C,(15)N]-labeled proteins have to be used in this type of studies.

Inhibitors of cancer cell multidrug resistance mediated by breast cancer resistance protein (BCRP/ABCG2).

Breast cancer resistance protein (BCRP/ABCG2) belongs to the ATP-binding cassette (ABC) transporter superfamily. It is able to efflux a broad range of anti-cancer drugs through the cellular membrane, thus limiting their anti-proliferative effects. Due to its relatively recent discovery in 1998, and in contrast to the other ABC transporters P-glycoprotein (MDR1/ABCB1) and multidrug resistance-associated protein (MRP1/ABCC1), only a few BCRP inhibitors have been reported.