1,720,993 research outputs found
Method for the preparation of surfactant peptides
No full textMethod for the preparation of surfactant-protein C peptides (SP-C peptides) based on heterologous expression of a fusion protein of an SP-C peptide and a maltose binding protein; genetic constructs, vectors and host cells for use in this method
Tryptophan synthase: a mine for enzymologists
No full textTryptophan synthase is a pyridoxal 5'-phosphate-dependent alpha(2)beta(2) complex catalyzing the last two steps of tryptophan biosynthesis in bacteria, plants and fungi. Structural, dynamic and functional studies, carried out over more than 40 years, have unveiled that: (1) alpha- and beta-active sites are separated by about 20 A and communicate via the selective stabilization of distinct conformational states, triggered by the chemical nature of individual catalytic intermediates and by allosteric ligands; (2) indole, formed at alpha-active site, is intramolecularly channeled to the beta-active site; and (3) naturally occurring as well as genetically generated mutants have allowed to pinpoint functional and regulatory roles for several individual amino acids. These key features have made tryptophan synthase a text-book case for the understanding of the interplay between chemistry and conformational energy landscapes
Identification of the Geometric Requirements for the Allosteric Communication between Alpha and Beta Subunits of Tryptophan Synthase
No full textThe pyridoxal 5'-phosphate-dependent tryptophan synthase alpha(2)beta(2) complex is a paradigmatic protein for substrate channeling and allosteric regulation. The enzymatic activity is modulated by a ligand-mediated equilibrium between open (inactive) and closed (active) conformations of the alpha- and beta-subunit, predominantly involving the mobile alpha loop 6 and the beta-COMM domain that contains beta helix 6. The alpha ligand-triggered intersubunit communication seems to rely on a single hydrogen bond formed between the carbonyl oxygen of beta Ser-178 of beta helix 6 and the NH group of alpha Gly-181 of alpha loop 6. We investigated whether and to what extent mutations of alpha Gly-181 and beta Ser-178 affect allosteric regulation by the replacement of beta Ser-178 with Pro or Ala and of alpha Gly-181 with either Pro to remove the amidic proton that forms the hydrogen bond or Ala, Val, and Phe to analyze the dependence on steric hindrance of the open-closed conformational transition. The alpha and beta activity assays and the equilibrium distribution of beta-subunit catalytic intermediates indicate that mutations do not significantly influence the intersubunit catalytic activation but completely abolish ligand-induced alpha- to beta-subunit signaling, demonstrating distinct pathways for alpha-beta-site communication. Limited proteolysis experiments indicate that the removal of the interaction between beta Ser-178 and alpha Gly-181 strongly favors the more trypsin-accessible open conformation of the alpha-active site. When the hydrogen bond cannot be formed, the alpha-subunit is unable to attain the closed conformation, and consequently, the allosteric signal is aborted at the subunit interface
Control of ionizable residues in the catalytic mechanism of tryptophan synthase from Salmonella typhimurium
No full textMethionine gamma lyase: Structure-activity relationships and therapeutic applications
No full text: Methionine gamma lyase (MGL) is a bacterial and plant enzyme that catalyzes the conversion of methionine in methanthiol, 2-oxobutanoate and ammonia. The enzyme belongs to fold type I of the pyridoxal 5'-dependent family. The catalytic mechanism and the structure of wild type MGL and variants were determined in the presence of the natural substrate as well as of many sulfur-containing derivatives. Structure-function relationship studies were pivotal for MGL exploitation in the treatment of cancer, bacterial infections, and other diseases. MGL administration to cancer cells leads to methionine starvation, thus decreasing cells viability and increasing their vulnerability towards other drugs. In antibiotic therapy, MGL acts by transforming prodrugs in powerful drugs. Numerous strategies have been pursued for the delivering of MGL in vivo to prolong its bioavailability and decrease its immunogenicity. These include conjugation with polyethylene glycol and encapsulation in synthetic or natural vesicles, eventually decorated with tumor targeting molecules, such as the natural phytoestrogens daidzein and genistein. The scientific achievements in studying MGL structure, function and perspective therapeutic applications came from the efforts of many talented scientists, among which late Tatyana Demidkina to whom we dedicate this review
Site-Specific Derivatization of Avidin Using Microbial Transglutaminase
No full textAvidin conjugates have several important applications in biotechnology and medicine. In this work, we investigated the possibility to produce site-specific derivatives of avidin using microbial transglutaminase (TGase). TGase allows the modification of proteins at the level of Gln or Lys residues using as substrate an alkyl-amine or a Gln-mimicking moiety, respectively. The reaction is site-specific, since Gln and Lys derivatization occurs preferentially at residues embedded in flexible regions of protein substrates. An analysis of the X-ray structure of avidin allowed us to predict Gln126 and Lys127 as potential sites of TGase's attack, because these residues are located in the flexible/unfolded C-terminal region of the protein. Surprisingly, incubation of avidin with TGase in the presence of alkylamine containing substrates (dansylcadaverine, 5-hydroxytryptamine) revealed a very low level of derivatization of the Gln126 residue. Analysis of the TGase reaction on synthetic peptide analogues of the C-terminal portion of avidin indicated that the lack of reactivity of Gln126 was likely due to the fact that this residue is proximal to negatively charged carboxylate groups, thus hampering the interaction of the substrate at the negatively charged active site of TGase. On the other hand, incubation of avidin with TGase in the presence of carbobenzoxy-l-glutaminyl-glycine in order to derivatize Lys residue(s) resulted in a clean and high yield production of an avidin derivative, retaining the biotin binding properties and the quaternary structure of the native protein. Proteolytic digestion of the modified protein, followed by mass spectrometry, allowed us to identify Lys127 as the major site of reaction, together with a minor modification of Lys58. By using TGase, avidin was also conjugated via a Lys-Gln isopeptide bond to a protein containing a single reactive Gln residue, namely, Gln126 of granulocyte-macrophage colony-stimulating factor. TGase can thus be exploited for the site-specific derivatization of avidin with small molecules or proteins
The molecular pathway for the allosteric regulation of tryptophan synthase
No full textThe pyridoxal 5'-phosphate (PLP)-dependent tryptophan synthase is a alpha(2)beta(2) complex. The alpha-beta subunit interaction plays a critical role both in the reciprocal activation of the individual subunits and in the allosteric regulation. We have investigated whether mutations of alphaloop6 Gly(181) and betahelix6 Ser(178) affect intersubunit communication. The loss of the hydrogen bond between these residues, achieved by proline substitution, does not significantly influence the intersubunit catalytic activation, but completely abolishes ligand-induced intersubunit signaling. The comparison of the crystal structure of the wild type and betaSer(178) Pro mutant, in the absence and presence of alpha-subunit ligands, indicates that the removal of the interaction between betaSer(178) and alphaGly(181) strongly affects the equilibrium between active (closed) and inactive (open) conformations of the a-active site, the latter being stabilized in both mutants
Allosteric communication between alpha and beta subunits of tryptophan synthase: modelling the open-closed transition of the alpha subunit
No full textLigand binding to the α-subunit of the α2β2 complex of tryptophan synthase induces the αloop6 closure over the α-active site. This conformational change is associated with the formation of a hydrogen bond between αGly181 NH group and βSer178 carbonyl oxygen, a key event for the triggering of intersubunit allosteric signals. Mutation of βSer178 to Pro and αGly181 to Pro, Ala, Phe and Val abolishes the ligand-induced intersubunit communication. Molecular dynamics methods were applied to simulate the conformation of the highly flexible and crystallographically undetectable open state of αloop6 in the wild type and in the α181 mutants. The open conformation of αloop6 is favoured in the wild type enzyme in the absence of α-ligands, and in the α181 mutants both in the presence and absence of bound ligands. A very good correlation was found between the extent of limited tryptic proteolysis and both the hydrogen bond distance between αX181 and βSer178, obtained from the molecular dynamics simulation, and the hydrogen bond strength, evaluated by HINT, an empirical force field that takes into account both enthalpic and entropic contributions. Comparison of the open and closed conformations of αloop6 suggests a pathway for substrate entrance into the α-active site and provides an explanation for the limited catalytic efficiency of the open state
ADIFAB fluorescence data used for the quantification of free fatty acids in media at different pH
No full textWe investigated the pH dependence of the fluorescence spectra of ADIFAB (FFA Sciences), a probe used for the quantification of free fatty acids (FFA). Data reports the change in the emission peak of ADIFAB and in the affinity for FFA as a function of pH. An algorithm based on spectral deconvolution allowed to correct ADIFAB fluorescence spectra for the spectroscopic effect caused by pH. Kd values were calculated at each pH based on a calibration with oleic acid. This method allows estimating FFA concentration by ADIFAB in media at different pH. The current data are related to the research article “Phospholipid components of the synthetic pulmonary surfactant CHF5633 probed by fluorescence spectroscopy” (Faggiano et al., 2018) [1]
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