Structural constraints for the Crh protein from solid-state NMR experiments

We demonstrate that short, medium and long-range constraints can be extracted from proton mediated, rare-spin detected correlation solid-state NMR experiments for the microcrystalline 10.4 × 2 kDa dimeric model protein Crh. Magnetization build-up curves from cross signals in NHHC and CHHC spectra deliver detailed information on side chain conformers and secondary structure for interactions between spin pairs. A large number of medium and long-range correlations can be observed in the spectra, and an analysis of the resolved signals reveals that the constraints cover the entire sequence, also including inter-monomer contacts between the two molecules forming the domain-swapped Crh dimer. Dynamic behavior is shown to have an impact on cross signals intensities, as indicated for mobile residues or regions by contacts predicted from the crystal structure, but absent in the spectra. Our work validates strategies involving proton distance measurements for large and complex proteins as the Crh dimer, and confirms the magnetization transfer properties previously described for small molecules in solid protein samples

Inverting family GH156 sialidases define an unusual catalytic motif for glycosidase action

Sialic acids are a family of related sugars that play essential roles in many biological events intimately linked to cellular recognition in both health and disease. Sialidases are therefore orchestrators of cellular biology and important therapeutic targets for viral infection. Here, we sought to define if uncharacterized sialidases would provide distinct paradigms in sialic acid biochemistry. We show that a recently discovered sialidase family, whose first member EnvSia156 was isolated from hot spring metagenomes, defines an unusual structural fold and active centre constellation, not previously described in sialidases. Consistent with an inverting mechanism, EnvSia156 reveals a His/Asp active center in which the His acts as a Bronsted acid and Asp as a Bronsted base in a single-displacement mechanism. A pre-dominantly hydrophobic aglycone site facilitates accommodation of a variety of 2-linked sialosides; a versatility that offers the potential for glycan hydrolysis across a range of biological and technological platforms

Coronin-1A Links Cytoskeleton Dynamics to TCRαβ-Induced Cell Signaling

Actin polymerization plays a critical role in activated T lymphocytes both in regulating T cell receptor (TCR)-induced immunological synapse (IS) formation and signaling. Using gene targeting, we demonstrate that the hematopoietic specific, actin- and Arp2/3 complex-binding protein coronin-1A contributes to both processes. Coronin-1A-deficient mice specifically showed alterations in terminal development and the survival of αβT cells, together with defects in cell activation and cytokine production following TCR triggering. The mutant T cells further displayed excessive accumulation yet reduced dynamics of F-actin and the WASP-Arp2/3 machinery at the IS, correlating with extended cell-cell contact. Cell signaling was also affected with the basal activation of the stress kinases sAPK/JNK1/2; and deficits in TCR-induced Ca2+ influx and phosphorylation and degradation of the inhibitor of NF-κB (IκB). Coronin-1A therefore links cytoskeleton plasticity with the functioning of discrete TCR signaling components. This function may be required to adjust TCR responses to selecting ligands accounting in part for the homeostasis defect that impacts αβT cells in coronin-1A deficient mice, with the exclusion of other lympho/hematopoietic lineages

CD and 1H-n.m.r. studies on the side-chain conformation of tyrosine derivatives and tyrosine residues in di- and tripeptides.

International audienceTyrosine, tyrosine peptides and derivatives, in total 11 species, were selected as models for the study of optical properties (1Lb band of phenolic group) and side-chain arrangement (rotamers around C alpha-C beta bond) of tyrosine as a function of chemical structure and pH effects. Circular dichroism spectra between 240 and 320 nm and n.m.r. spectra were recorded for the different ionization states. Results are discussed in terms of charge effects from N- and C-terminal groups and local conformation influence on 1Lb band of the phenolic chromophore and on distribution of rotamer populations in side-chain of tyrosine. Fractions of rotamer populations were estimated from alpha-beta proton-proton coupling constants and, in the cases of tyrosine and N-acetyl-tyrosine, from 15N-beta nitrogen-proton coupling constants, which allow the stereospecific assignment of the beta and beta' protons. The rotamer populations of tyrosine, averaged from all the data of the samples In solution, were then compared with their statistical distribution in th solid state. Interestingly, agreement is excellent when we refer to crystal of tyrosine, tyrosine derivatives or small peptides (31 samples) and poor in the case of proteins. This leads to a discussion on both the validity of using statistical distributions of rotamers in proteins as reference for rotamer preferences inside small peptides in solution and the choice of the appropriate Jg and Jt values in Pachler's approach. The possible existence of a correlation between ellipticity and rotamer populations for such samples is examined.Tyrosine, tyrosine peptides and derivatives, in total 11 species, were selected as models for the study of optical properties (1Lb band of phenolic group) and side-chain arrangement (rotamers around C alpha-C beta bond) of tyrosine as a function of chemical structure and pH effects. Circular dichroism spectra between 240 and 320 nm and n.m.r. spectra were recorded for the different ionization states. Results are discussed in terms of charge effects from N- and C-terminal groups and local conformation influence on 1Lb band of the phenolic chromophore and on distribution of rotamer populations in side-chain of tyrosine. Fractions of rotamer populations were estimated from alpha-beta proton-proton coupling constants and, in the cases of tyrosine and N-acetyl-tyrosine, from 15N-beta nitrogen-proton coupling constants, which allow the stereospecific assignment of the beta and beta' protons. The rotamer populations of tyrosine, averaged from all the data of the samples In solution, were then compared with their statistical distribution in th solid state. Interestingly, agreement is excellent when we refer to crystal of tyrosine, tyrosine derivatives or small peptides (31 samples) and poor in the case of proteins. This leads to a discussion on both the validity of using statistical distributions of rotamers in proteins as reference for rotamer preferences inside small peptides in solution and the choice of the appropriate Jg and Jt values in Pachler's approach. The possible existence of a correlation between ellipticity and rotamer populations for such samples is examined

Estudio de la producción de ácido itacónico con Aspergillus terreus de la cepa MJL05 con diferentes variables

La producción de ácido itacónico (AI) con Aspergillus terreus MJL05 se realizó en fermentación sumergida en lote en un biorreactor agitado para estudiar el efecto de la variación de las concentraciones de nitrógeno, fósforo y carbono en el medio de producción. El glicerol, subproducto del biodiesel, fue reportado como un sustrato eficiente para obtener altas productividades de AI. Este fue utilizado como única fuente de carbono. Las relaciones entre nutrientes, C:N 18, N:P 10,8 y C:P 195 fueron seleccionadas como las mejores para aumentar la concentración de AI de 11,0 a 27,6 g/l con un a productividad volumétrica de 0,192 g IA/l.h, y una productividad específica de 0,013 g IA/g biomasa.h. Los rendimientos del bioproceso obtenidos fueron de Yx:s 0,27 g p.s. biomasa/g sustrato; Yp:x 1,63 g IA/g p.s. biomasa y Yp:s 0,44 g IA/g sustrato, lo que permite asumir la factibilidad de usar esta cepa para la producción de AI.Itaconic acid (IA) production by Aspergillus terreus MJL05 strain was investigated in submerged batch fermentation in a stirred bioreactor to determine the effect of varying the nitrogen, phosphorous and carbon sources concentrations in the production medium. Glycerol, a biodiesel by-product was reported as an efficient substrate to achieve high itaconic acid productivities. This was used as the sole carbon source. The resulting C:N 18, N:P 10.8 and C:P 195 ratios were selected as the best and allowed to improve IA concentration from 11.0 to 27.6 g/l with a volumetric productivity of 0.192 g IA/l.h and a specific productivity 0.013 g IA/g biomass.h. Bioprocess yields, Yx:s 0.27 g d.w. biomass/g substrate; Yp:x 1.63 g IA/g d.w. biomass and Yp:s 0.44 g IA/g substrate, allowed to assume the feasibility of using this strain for IA production

Conformation and mobility of tyrosine side chain in tetrapeptides. Specific effects of cis- and trans-proline in Tyr-Pro- and Pro-Tyr-segments.

International audienceWe examined the properties of tyrosine in four free tetrapeptides: Ala-Ala-Tyr-Ala (AATA), Ala-Pro-Tyr-Ala (APTA), Ala-Tyr-Ala-Ala (ATAA) and Ala-Tyr-Pro-Ala (ATPA) by CD, n.m.r. and energy calculations. Experimental data (the aromatic 1Lb signal, rotamer populations around the C alpha-C beta bond (chi 1), rotations around C beta-C gamma(chi 2), chemical shifts of ortho- and meta-protons in the phenolic ring (in aqueous and Me2SO solutions), NH proton temperature coefficients and vicinal coupling constants 3JNH-C alpha H in the backbone (Me2SO solution) were compared with calculated minimum energy conformations. We find qualitative agreement between the results of the different techniques with respect to global tendencies of conformational behaviour: we present experimental evidence showing that the presence of proline in the sequence has a more pronounced effect on the side chain organization of the residues preceding it than on one succeeding it. This steric influence of proline on its immediate neighbor is even stronger in the cis isomer than in the more common trans isomer. The strong preference for Rotamer II (chi 1 = 180 degrees) over Rotamer I (chi 1 = -60 degrees) in ATPA (cis-form) concomitant with a noticeable deviation of chi 2 is a striking example.We examined the properties of tyrosine in four free tetrapeptides: Ala-Ala-Tyr-Ala (AATA), Ala-Pro-Tyr-Ala (APTA), Ala-Tyr-Ala-Ala (ATAA) and Ala-Tyr-Pro-Ala (ATPA) by CD, n.m.r. and energy calculations. Experimental data (the aromatic 1Lb signal, rotamer populations around the C alpha-C beta bond (chi 1), rotations around C beta-C gamma(chi 2), chemical shifts of ortho- and meta-protons in the phenolic ring (in aqueous and Me2SO solutions), NH proton temperature coefficients and vicinal coupling constants 3JNH-C alpha H in the backbone (Me2SO solution) were compared with calculated minimum energy conformations. We find qualitative agreement between the results of the different techniques with respect to global tendencies of conformational behaviour: we present experimental evidence showing that the presence of proline in the sequence has a more pronounced effect on the side chain organization of the residues preceding it than on one succeeding it. This steric influence of proline on its immediate neighbor is even stronger in the cis isomer than in the more common trans isomer. The strong preference for Rotamer II (chi 1 = 180 degrees) over Rotamer I (chi 1 = -60 degrees) in ATPA (cis-form) concomitant with a noticeable deviation of chi 2 is a striking example

Amylose chain behavior in an interacting context .1. Influence of a nonchair ring on the maltose conformations

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Amylose chain behavior in an interacting context II. Molecular modeling of a maltopentaose fragment in the barley alpha-amylase catalytic site

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