Development and optimization of a binding assay for histone deacetylase 4 using surface plasmon resonance

Electroelution is a widely used methodology for protein purification. In this study, a practical and low-cost system for protein electroelution from stained polyacrylamide gels was developed. For this, a horizontal protein electroelution cuve was constructed with glass plates, 1.5-ml capacity microcentrifuge tubes, and dialysis membrane. Analyses of the system efficiency showed high protein recovery from nonfixed and fixed sodium dodecyl sulfate (SDS)-polyacrylamide gels.     

Bio-Based Succinate Production from <Emphasis Type="Italic">Arundo donax</Emphasis> Hydrolysate with the New Natural Succinic Acid-Producing Strain <Emphasis Type="Italic">Basfia succiniciproducens</Emphasis> BPP7

Bio-based succinic acid production from lignocellulosic biomass is one of the attractive and prominent alternative technologies to overcome issues associated with the utilization of fossil sources. In this context, it is necessary to find new microorganisms that are able to efficiently ferment this recalcitrant feedstock. The ecological approach developed in this study enabled the isolation of Basfia succiniciproducens BPP7 from a complex rumen ecosystem. This new wild-type strain was able to synthesize up to 6.06 ± 0.05 g/L of succinate (corresponding to 0.84 ± 0.017 g of succinate per gram of consumed glucose + xylose and to 0.14 ± 0.001 g of succinate per gram of glucans + xylans present in the biomass before hydrolysis) from Arundo donax hydrolysate in separate hydrolysis and fermentation (SHF) experiments. Higher titers of succinic acid were obtained through the optimization of growth conditions. The optimal medium composition identified on the smaller scale was then used for 2.5-L batch experiments, which used A. donax hydrolysate and yeast extract as the main C and N sources, respectively. A maximal titer of 9.4 ± 0.4 g/L of succinic acid was obtained after 24 h. The overall results clearly demonstrate the potential of B. succiniciproducens BPP7 for succinate production.     

2-Trifluoroacetylthiophenes, a novel series of potent and selective class II histone deacetylase inhibitors

The identification of class II HDAC inhibitors has been hampered by lack of efficient enzyme assays, in the preceding paper two assays have been developed to improve the efficiency of these enzymes: mutating an active site histidine to tyrosine, or by the use of a trifluoroacetamide lysine substrate, allowing screening to identify class II HDAC inhibitors. Herein, 2-trifluoroacetylthiophenes have been demonstrated to inhibit class II HDACs, resulting in the development of a series of 5-(trifluoroacetyl)thiophene-2-carboxamides as novel, potent and selective class II HDAC inhibitors. X-ray crystal structures of the HDAC 4 catalytic domain with a bound inhibitor demonstrate these compounds are active site inhibitors and bind in their hydrated form.     

Optimization of a series of potent and selective ketone histone deacetylase inhibitors

Histone deacetylase (HDAC) inhibitors offer a promising strategy for cancer therapy and the first generation HDAC inhibitors are currently in the clinic. Herein we describe the optimization of a series of ketone small molecule HDAC inhibitors leading to potent and selective class I HDAC inhibitors with good dog PK.     

Structure and function correlations between the rat liver threonine deaminase and aminotransferases

The rat liver threonine deaminase is a cytoplasmic enzyme that catalyses the pyridoxal-phosphate-dependent dehydrative deamination of L-threonine and L-serine to ammonia and alpha-ketobutyrate and pyruvate, respectively, in vivo. During deamination, a molecule of the cofactor is converted to pyridoxamine phosphate. Recently, the ability of this enzyme to accomplish an inverse half-reaction, restoring pyridoxal-phosphate and L-alanine or L-aminobutyrate, respectively, from pyruvate or 2-oxobutyrate, was reported. In order to investigate the molecular mechanisms of this transaminating activity, a molecular model of rat liver threonine deaminase was constructed on the basis of sequence homology with the biosynthetic threonine deaminase of Escherichia coli, the crystal structure of which is known. The model has structural features shared by aminotransferases, suggesting that tertiary structural elements may be responsible for the transaminating activity observed for rat liver threonine deaminase.     

Peptide-protein interactions studied by surface plasmon and nuclear magnetic resonances

The structural features of the complexes that alpha-bungarotoxin forms with three different synthetic peptides, mimotopes of the nicotinic acetylcholine receptor binding site, have been compared to the corresponding nuclear magnetic resonance (NMR) and surface plasmon resonance (SPR) data. For the considered peptides, the observed different affinities towards the toxin could not be accounted simply by static structural considerations. A combined analysis of the SPR- and NMR-derived dynamic parameters shows new correlations between complex formation and dissociation and the overall pattern of intramolecular and intermolecular nuclear Overhauser effects. These features could be crucial for a rational design of protein ligands.     

NMR structure of alpha-bungarotoxin free and bound to a mimotope of the nicotinic acetylcholine receptor

A combinatorial library approach was used to produce synthetic peptides mimicking the snake neurotoxin binding site of nicotinic receptors. Among the sequences, which inhibited binding of alpha-bungarotoxin to muscle and neuronal nicotinic receptors, HRYYESSLPWYPD, a 14-amino acid peptide with considerably higher toxin-binding affinity than the other synthesized peptides, was selected, and the structure of its complex with the toxin was analyzed by NMR. Comparison of the solution structure of the free toxin and its complex with this peptide indicated that complex formation induced extensive conformational rearrangements mainly at finger II and the carboxy terminus of the protein. The peptidyl residues P10 and Y4 seemed to be critical for peptide folding and complex stability, respectively. The latter residue of the peptide strongly interacted with the protein by entering a small pocket delimited by D30, C33, S34, R36, and V39 toxin side chains.     

NMR studies of protein hydration and TEMPOL accessibility

Understanding the mechanisms of the interaction between a protein surface and its outer molecular environment is of primary relevance for the rational design of new drugs and engineered proteins. Protein surface accessibility is emerging as a new dimension of Structural Biology, since NMR methods have been developed to follow how molecules, even those different from physiological ligands, preferentially approach specific regions of the protein surface. Hen egg-white lysozyme, a paradigmatic example of the state of the art of protein structure and dynamics, has been selected as a model system to study protein surface accessibility. Bound water and soluble spin-labels have been used to investigate the interaction of this enzyme, both free and bound to the inhibitor (NAG)(3), with its molecular environment. No tightly bound water molecules were found inside the enzyme active site, which, conversely, appeared as the most exposed to visits from the soluble paramagnetic probe TEMPOL. From the presented set of data, an integrated view of lysozyme surface accessibility towards water and TEMPOL molecules is obtained.