Effect of size and N-terminal residue characteristics on bacterial cell penetration and antibacterial activity of the proline-rich peptide Bac7

Bac7 is a proline-rich antimicrobial peptide, selective for Gram-negative bacteria, which acts intracellularly after membrane translocation. Progressively shortened fragments of Bac7 allowed determining the minimal sequence required for entry and antimicrobial activity as a 16-residue, N-terminal fragment, while further shortening led to a marked decrease in both functions. Furthermore, two N-terminal arginine residues were required for efficient translocation and activity. Analogues in which these residues were omitted, or where the side chain steric or physicochemical characteristics were systematically altered, were tested on different Escherichia coli strains, including a mutant with a destabilized outer membrane and one lacking the relevant SbmA membrane transport protein. H-bonding capacity, stereochemistry, and charge, in that order, played a determining role for efficient transit through both the outer and cytoplasmic membranes. Our studies allowed building a more detailed model for the mode-of-action of Bac7, and confirming its potential as an anti-infective agent, also suggesting it may be a vehicle for internalization of other antibiotic cargo

C–H insertion as a key step to spiro-oxetanes, scaffolds for drug discovery

A new route to spiro-oxetanes, potential scaffolds for drug discovery, is described. The route is based on the selective 1,4-C–H insertion reactions of metallocarbenes, generated from simple carbonyl precursors in flow or batch mode, to give spiro-β-lactones that are rapidly converted into spiro-oxetanes. The three-dimensional and lead like-properties of spiro-oxetanes is illustrated by the conversion of the 1-oxa-7-azaspiro[3,5]nonane scaffold into a range of functionalized derivatives

Novel Protocol for the Chemical Synthesis of Crustacean Hyperglycemic Hormone Analogues — An Efficient Experimental Tool for Studying Their Functions

The crustacean Hyperglycemic Hormone (cHH) is present in many decapods in different isoforms, whose specific biological functions are still poorly understood. Here we report on the first chemical synthesis of three distinct isoforms of the cHH of Astacus leptodactylus carried out by solid phase peptide synthesis coupled to native chemical ligation. The synthetic 72 amino acid long peptide amides, containing L- or D-Phe3 and (Glp1, D-Phe3) were tested for their biological activity by means of homologous in vivo bioassays. The hyperglycemic activity of the D-isoforms was significantly higher than that of the L-isoform, while the presence of the N-terminal Glp residue had no influence on the peptide activity. The results show that the presence of D-Phe3 modifies the cHH functionality, contributing to the diversification of the hormone pool

Oxidative folding of Amaranthus alpha-amylase inhibitor: disulfide bond formation and conformational folding.

Oxidative folding is the fusion of native disulfide bond formation with conformational folding. This complex process is guided by two types of interactions: first, covalent interactions between cysteine residues, which transform into native disulfide bridges, and second, non-covalent interactions giving rise to secondary and tertiary protein structure. The aim of this work is to understand both types of interactions in the oxidative folding of Amaranthus alpha-amylase inhibitor (AAI) by providing information both at the level of individual disulfide species and at the level of amino acid residue conformation. The cystine-knot disulfides of AAI protein are stabilized in an interdependent manner, and the oxidative folding is characterized by a high heterogeneity of one-, two-, and three-disulfide intermediates. The formation of the most abundant species, the main folding intermediate, is favored over other species even in the absence of non-covalent sequential preferences. Time-resolved NMR and photochemically induced dynamic nuclear polarization spectroscopies were used to follow the oxidative folding at the level of amino acid residue conformation. Because this is the first time that a complete oxidative folding process has been monitored with these two techniques, their results were compared with those obtained at the level of an individual disulfide species. The techniques proved to be valuable for the study of conformational developments and aromatic accessibility changes along oxidative folding pathways. A detailed picture of the oxidative folding of AAI provides a model study that combines different biochemical and biophysical techniques for a fuller understanding of a complex process

DNA-mediated assembly of weakly interacting DNA-binding protein subunits: in vitro recruitment of phage 434 repressor and yeast GCN4 DNA-binding domains

The specificity of DNA-mediated protein assembly was studied in two in vitro systems, based on (i) the DNA-binding domain of bacteriophage 434 repressor cI (amino acid residues 1–69), or (ii) the DNA-binding domain of the yeast transcription factor GCN4, (amino acids 1–34) and their respective oligonucleotide cognates. In vivo, both of these peptides are part of larger protein molecules that also contain dimerization domains, and the resulting dimers recognize cognate palindromic DNA sequences that contain two half-sites of 4 bp each. The dimerization domains were not included in the peptides tested, so in solution—in the presence or absence of non-cognate DNA oligonucleotides—these molecules did not show appreciable dimerization, as determined by pyrene excimer fluorescence spectroscopy and oxidative cross-linking monitored by mass spectrometry. Oligonucleotides with only one 4 bp cognate half-site were able to initiate measurable dimerization, and two half-sites were able to select specific dimers even from a heterogeneous pool of molecules of closely related specificity (such as DNA-binding domains of the 434 repressor and their engineered mutants that mimic the binding helix of the related P22 phage repressor). The fluorescent technique allowed us to separately monitor the unspecific, ionic interaction of the peptides with DNA which produced a roughly similar signal in the case of both cognate and non-cognate oligonucleotides. But in the former case, a concomitant excimer fluorescence signal showed the formation of correctly positioned dimers. The results suggest that DNA acts as a highly specific template for the recruitment of weakly interacting protein molecules that can thus build up highly specific complexes

Arginine methylation of a mitochondrial guide RNA binding protein from Trypanosoma brucei

RBP16 is a mitochondrial Y-box protein from the parasitic protozoan Trypanosoma brucei that binds guide RNAs and ribosomal RNAs. It is comprised of an N-terminal cold-shock domain and a C-terminal domain rich in glycine and arginine residues, resembling the RGG RNA-binding motif. Arginine residues found within RGG domains are frequently asymmetrically dimethylated by a class of enzymes termed protein arginine methyltransferases (PRMTs). As Arg-93 of RBP16 exists in the context of a preferred sequence for asymmetric arginine dimethylation (G/FGGRGGG/F), we investigated whether modified arginines are present in native RBP16 by MALDI-TOF and post-source decay analyses. These analyses confirmed that Arg-93 is dimethylated. In addition, Arg-78 exists as an unmodified or as a monomethylated derivative, and Arg-85 is present in forms corresponding to the unmodified, di-, and tri-methylated state. While Arg-93 is apparently constitutively dimethylated, the methylation of Arg-78 and Arg-85 is mutually exclusive. Furthermore, whole cell extracts from procyclic form T. brucei are able to methylate bacterially expressed RBP16 (rRBP16), as well as endogenous proteins, in the presence of S-adenosyl- l-[ methyl- 3H]methionine. While assays of mitochondrial extracts suggest a small amount of PRMT may also be present in this subcellular compartment, the majority of trypanosome PRMT activity is extramitochondrial. We show that rRBP16 is methylated in trypanosome extracts through the action of a type I methyltransferase as well as serving as a substrate for heterologous mammalian type I PRMTs. In addition, we demonstrate the presence of type II PRMT activity in trypanosome cell extracts. These results suggest that protein arginine methylation is a common posttranslational modification in trypanosomes, and that it may regulate the function of RBP16

Oxidative folding intermediates with nonnative disulfide bridges between adjacent cysteine residues

The oxidative folding of the Amaranthus α-amylase inhibitor, a 32-residue cystine-knot protein with three disulfide bridges, was studied in vitro in terms of the disulfide content of the intermediate species. A nonnative vicinal disulfide bridge between cysteine residues 17 and 18 was found in three of five fully oxidized intermediates. One of these, the most abundant folding intermediate (MFI), was further analyzed by (1)H NMR spectroscopy and photochemically induced dynamic nuclear polarization, which revealed that it has a compact structure comprising slowly interconverting conformations in which some of the amino acid side chains are ordered. NMR pulsed-field gradient diffusion experiments confirmed that its hydrodynamic radius is indistinguishable from that of the native protein. Molecular modeling suggested that the eight-membered ring of the vicinal disulfide bridge in MFI may be located in a loop region very similar to those found in experimentally determined 3D structures of other proteins. We suggest that the structural constraints imposed on the folding intermediates by the nonnative disulfides, including the vicinal bridge, may play a role in directing the folding process by creating a compact fold and bringing the cysteine residues into close proximity, thus facilitating reshuffling to native disulfide bridges

Time course of the induced glycemia after injection of the synthetic and wild type cHHs.

<p>a) Time course of hemolymph glycemia after injection of 1.7 pmol/g live weight of D-cHH. The maximum peak of 95.4±8.1 mg/dL glucose is reached after 4 h, high values lasting at 8 h (76±12.1 mg/dL), when the maximum values were also recorded, and the glycemia returning to its nearby basal level, 8.9±1 mg/dL, after 24 h. N = 20. b) Time course of hemolymph glycemia after injection of 1.7 pmol/g live weight of L-cHH. L-cHH induced a slower hyperglycemic response with the maximum peak of 52±6.5 mg/dL glucose after 2 h, and the glycemia returning to its nearby basal level, 5.5±0.4 mg/dL, after 8 h. N = 16. c) Time course of hemolymph glycemia after injection of 1.7 pmol/g live weight of Glp-D-cHH. The time course of Glp-D-cHH shows the stronger hyperglycemic response of 96.1±5.6 mg/dL glucose after 4 h, high values lasting at 8 h (62.9±11.8 mg/dL) and the glycemia returning to its basal level, 2.9±0.5 mg/dL, after 24 h. N = 9. d) Time course of hemolymph glycemia after injection of 1.7 pmol/g live weight of SG-cHH. The hyperglycemic time course after the injection of 1.7 pmol/g live weight of purified SG-cHH was comparable to that of synthetic D-cHH and Glp-D-cHH peptides, but the maximum (44±4.5 mg/dL) was reached after 2 h and the glycemia returning to its basal level, 5±1.1 mg/dL after 24 h. N = 9.</p

Boxplots of the maximum glucose values recorded for the synthetic and native peptides.

<p>Both D-cHH and Glp-D-cHH induced a strong hyperglycemia with a mean peak glucose concentration of, respectively, 100.5±9.3 and 98.7±6 mg/dL, while the hyperglycemic effect of GS-cHH and of L-cHH was lower, with a mean of max glycemia of 52.7±9.4 mg/dL and of 52.4±6.4 mg/dL respectively.</p

Overview of insulin production and purification process from <i>Pichia pastoris</i> fermentation culture.

<p>Overview of insulin production and purification process from <i>Pichia pastoris</i> fermentation culture.</p