Clovis R. Nakaie

Internally quenched fluorogenic protease substrates: Solid-phase synthesis and fluorescence spectroscopy of peptides containing ortho-aminobenzoyl/dinitrophenyl groups as donor-acceptor pairs

A general procedure, using the commonly employed solid-phase peptide synthesis methodology for obtaining internally quenched fluorogenic peptides with ortho-aminobenzoyl/dinitrophenyl groups as donor-acceptor pairs, is presented. The essential feature of this procedure is the synthesis of an N α-Boc or-Fmoc derivative of glutamic acid with the α-carboxyl group bound to N-(2,4-dinitrophenyl)-ethylenediamine (EDDnp), which provides the quencher moiety attached to the C-terminus of the substrate. The fluorescent donor group, ortho-aminobenzoic acid (Abz), is incorporated into the resin-bound peptide in the last coupling cycle. Depending on the resin type used, Abz-peptidyl-Gln-EDDnp or Abz-peptidyl-Glu-EDDnp is obtained. Using the procedure described above, substrates for human renin and tissue kallikreins were synthesised. Spectrofluorimetric measurements of Abz bound to the α-amino group of proline showed that strong quenching of Abz fluorescence occurs in the absence of any acceptor group.

Solvation of polymers as model for solvent effect investigation: proposition of a novel polarity scale

Abstract A precise understanding of the polymer solvation effect has been considered crucial to many modern methods, but its dependence on the polarity of the medium is still not entirely established. To more thoroughly address this issue, the swelling degrees of polymers with a great variety of structures, taken as solute-models, were measured and correlated with the polarity of ca. 30 solvent systems. Relevant for any resin-supported methods, a characteristic solvation behavior of each class of polymeric material was detected. Moreover by interpreting the relationship between the large set of solute–solvent interaction data and the most solvent properties known so far, the sum of solvent electron acceptor (AN) and donor (DN) numbers, at a 1:1 proportion was suggested as an alternative and more accurate empirical solvent polarity scale.

Synthesis and properties of spin-labeled angiotensin derivatives

Abstract We have synthesized three derivatives of the peptide hormone angiotensin, containing as their N-terminal residue the spin-labeled amino acid 2,2,6,6-tetramethylpiperidine-N-oxide-4-amino-4-carboxylicacid. (TOAC). The angiotensin analogs displayed considerable biological activity, indicating that the spin label is not too great a perturbation for the hormone-receptor interaction. Studies of the effect of pH upon the electron paramagnetic resonance (EPR) spectra of the spin-labeled angiotensins indicated that deprotonation of the terminal amino group leads to changes in spectral parameters similar to those displayed by model compounds (TOAC and TOAC-glycine). In view of the slow exchange between the two forms at pH values where both the protonated and unprotonated forms coexist in considerable amounts, computer simulations demonstrate that the EPR spectra are superpositions of the spectra for each form. The EPR spectra of the spin-labeled hormone derivatives were shown to be indicative of a pH-dependent conformationai change, corroborating previous conclusions drawn from other studies. This study demonstrates the usefulness of spin-labeled analogs for the investigation of conformational properties of small peptides.

Bradykinin-related peptides from Phyllomedusa hypochondrialis

Bradykinin related peptides (BRPs) present in the water-soluble secretion and freshly dissected skin fragments of Phyllomedusa hypochondrialis were investigated by mass spectrometry techniques. Eighteen BRPs, along with their post-translational modifications, were characterized in the secretion by de novo MS/MS sequencing and direct MALDI imaging experiments of the frog skin. These molecules revealed strong sequence similarities to the main plasma kinin of some mammals and reptiles. Such a diversity of molecules, within the same peptide family, belonging to a single amphibian species may be related to functional specializations of these peptides and a variety of corresponding receptors that might be present in a number of different predators. Also, a novel analog, [Val]1,[Thr]6-bradykinyl-Gln,Ser had its biological activity positively detected in cell culture expressing the human bradykinin B2 receptor and in guinea pig ileum preparations.

First synthesis of a fully active spin-labeled peptide hormone

For the first time in the electron spin resonance (ESR) and peptide synthesis fields, a fully active spin‐labeled peptide hormone was reported. The ESR spectra of this α‐melanocyte stimulating hormone (α‐MSH) analogue (acetyl‐Toac0‐α‐MSH) where Toac is the paramagnetic amino acid probe 2,2,6,6‐tetramethylpiperidine‐1‐oxyl‐4‐amino‐4‐carboxylic acid, suggested a pH‐independent conformation and a more restricted movement comparatively to the free Toac. Owing to its equivalent biological potency in a skin pigmentation assay as compared to the native α‐MSH and its unique characteristic (paramagnetic, naturally fluorescent and fully active), this analogue is of great potential for investigation of relevant physiological roles reported for α‐MSH.

Use of spin label EPR spectra to monitor peptide chain aggregation inside resin beads

Abstract An EPR approach to monitor peptide chain aggregation inside resin beads is introduced. Model low and highly peptide-loaded resins containing an aggregating sequence were labeled with a paramagnetic amino acid derivative and studied with regard to their solvation behavior in different solvent systems. For the first time in the peptide synthesis, EPR spectroscopic has allowed the detection of differentiated levels of peptide chain aggregation as a function of solvent and resin loading.

Synthesis and pharmacological properties of TOAC-labeled angiotensin and bradykinin analogs

Angiotensin II (AngII) and bradykinin (BK) derivatives containing the TOAC (2,2,6,6-tetramethylpiperidine-N-oxyl-4-amino-4-carboxylic acid) spin label were synthesized by solid phase methodology. Ammonium hydroxide (pH 10, 50 degrees C, l h) was the best means for reverting nitroxide protonation occurring during peptide cleavage. EPR spectra yielded rotational correlation times for internally labeled analogs that were nearly twice as large as those of N-terminally labeled analogs. Except for TOAC(1)-AngII and TOAC(0)-BK, which showed high intrinsic activities, other derivatives were inactive in smooth muscle preparations. These active paramagnetic analogs may be useful for conformational studies in solution and in the presence of model and biological membranes.

Synthesis and immunological activity of a branched peptide carrying the T-cell epitope of gp43, the major exocellular antigen of Paracoccidioides brasiliensis

The 43 kDa glycoprotein (gp43) of Paracoccidioides brasiliensis is the major diagnostic antigen of paracoccidioidomycosis (PCM), a prevalent fungal infection in South America. A 15‐mer sequence from gp43, denominated P10, induced T‐CD4+ T helper 1 cellular immune responses in mice of three different haplotypes and protected against intratracheal challenge by a virulent isolate of P. brasiliensis. In an attempt to improve delivery of P10, a promiscuous antigen also presented by human leucocyte antigen‐DR alleles, aiming at immunotherapy, we synthesized a multiple antigen peptide with the protective T‐cell epitope expressed in a tetravalent 13‐mer analog of P10 (M10). M10 induced specific lymph node cell proliferation in mice preimmunized with peptides in complete Freunds adjuvant (CFA). In addition, M10 immunization without CFA significantly protected intratracheally infected mice. We conclude that M10 is a candidate for an anti‐PCM vaccine. In this report we describe: (1) the synthesis of M10; (2) the induction of M10‐elicited T‐cell response and (3) in vivo protection of mice immunized with M10 and challenged by a virulent strain of P. brasiliensis.

Structure of Two Fragments of the Third Cytoplasmic Loop of the Rat Angiotensin II AT1A Receptor IMPLICATIONS WITH RESPECT TO RECEPTOR ACTIVATION AND G-PROTEIN SELECTION AND COUPLING

The structural bases that render the third intracellular loop (i3) of the rat angiotensin II AT1Areceptor one of the cytoplasmic domains responsible for G-protein coupling are still unknown. The three-dimensional structures of two overlapping peptides mapping the entire i3 loop and shown to differently interact with purified G-proteins have been obtained by simulated annealing calculations, using NMR-derived constraints collected in 70% water/30% trifluoroethanol solution. While the NH2-terminal half, Ni3, residues 213–231, adopts a stable amphipathic α-helix, extending over almost the entire peptide, a more flexible conformation is found for the COOH-terminal half, Ci3, residues 227–242. For this peptide, a cis-transisomerization around the Lys6—Pro7 peptide bond generates two exchanging isomers adopting similar conformations, with an α-helix spanning from Asn9 to Ile15and a poorly defined NH2 terminus. A quite distinct structural organization is found for the sequence EIQKN, common to Ni3 and Ci3. The data do suggest that the extension and orientation of the amphipathic α-helix, present in the proximal part of i3, may be modulated by the distal part of the loop itself through the Pro233 residue. A molecular model where this possibility is considered as a mechanism for G-protein selection and coupling is presented.

Ion channel-like activity of the antimicrobial peptide tritrpticin in planar lipid bilayers.

The cationic peptide tritrpticin (VRRFPWWWPFLRR, Trp3) has a broad action spectrum, acting against Gram‐positive and Gram‐negative bacteria, as well as some fungi, while also displaying hemolytic activity. We have studied the behavior of Trp3 in planar lipid bilayers (or black lipid membrane – BLM) and were able to demonstrate its ion channel‐like activity. Channel‐like activity was observed in negatively charged azolectin BLM as a sudden appearance of discrete current fluctuations upon application of a constant voltage across the membrane. Trp3 formed large conductance channels (500–2000 pS) both at positive and negative potentials. In azolectin bilayers, the predominant ion‐channel activity was characterized by very regular and discrete current steps (corresponding to openings) of uniform amplitude, which exhibited relatively long residence times (of the order of seconds). Occasionally, multiple conductance steps were observed, indicating the simultaneous presence of more than one open pore. In bilayers of zwitterionic diphytanoylphosphatidyl choline (DPhPC) Trp3 also showed ion‐channel activity, but in a much less frequent and less prominent way. Studies of ion selectivity indicated that Trp3 forms a cation‐selective channel. These results should contribute to the understanding of the molecular interactions and mechanism of action of Trp3 in lipid bilayers and biological membranes.