Short peptide constructs mimic agonist sites of AT(1)R and BK receptors.

Extracellular peptide ligand binding sites, which bind the N-termini of angiotensin II (AngII) and bradykinin (BK) peptides, are located on the N-terminal and extracellular loop 3 regions of the AT(1)R and BKRB(1) or BKRB(2) G-protein-coupled receptors (GPCRs). Here we synthesized peptides P15 and P13 corresponding to these receptor fragments and showed that only constructs in which these peptides were linked by S-S bond, and cyclized by closing the gap between them, could bind agonists. The formation of construct-agonist complexes was revealed by electron paramagnetic resonance spectra and fluorescence measurements of spin labeled biologically active analogs of AngII and BK (Toac(1)-AngII and Toac(0)-BK), where Toac is the amino acid-type paramagnetic and fluorescence quencher 2, 2, 6, 6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid. The inactive derivatives Toac(3)-AngII and Toac(3)-BK were used as controls. The interactions characterized by a significant immobilization of Toac and quenching of fluorescence in complexes between agonists and cyclic constructs were specific for each system of peptide-receptor construct assayed since no crossed reactions or reaction with inactive peptides could be detected. Similarities among AT, BKR, and chemokine receptors were identified, thus resulting in a configuration for AT(1)R and BKRB cyclic constructs based on the structure of the CXCR(4), an α-chemokine GPCR-type receptor.

The spin label amino acid TOAC and its uses in studies of peptides: chemical, physicochemical, spectroscopic, and conformational aspects.

We review work on the paramagnetic amino acid 2,2,6,6-tetramethyl-N-oxyl-4-amino-4-carboxylic acid, TOAC, and its applications in studies of peptides and peptide synthesis. TOAC was the first spin label probe incorporated in peptides by means of a peptide bond. In view of the rigid character of this cyclic molecule and its attachment to the peptide backbone via a peptide bond, TOAC incorporation has been very useful to analyze backbone dynamics and peptide secondary structure. Many of these studies were performed making use of EPR spectroscopy, but other physical techniques, such as X-ray crystallography, CD, fluorescence, NMR, and FT-IR, have been employed. The use of double-labeled synthetic peptides has allowed the investigation of their secondary structure. A large number of studies have focused on the interaction of peptides, both synthetic and biologically active, with membranes. In the latter case, work has been reported on ligands and fragments of GPCR, host defense peptides, phospholamban, and ß-amyloid. EPR studies of macroscopically aligned samples have provided information on the orientation of peptides in membranes. More recent studies have focused on peptide-protein and peptide-nucleic acid interactions. Moreover, TOAC has been shown to be a valuable probe for paramagnetic relaxation enhancement NMR studies of the interaction of labeled peptides with proteins. The growth of the number of TOAC-related publications suggests that this unnatural amino acid will find increasing applications in the future.

Conformational properties of angiotensin II and its active and inactive TOAC-labeled analogs in the presence of micelles. Electron paramagnetic resonance, fluorescence, and circular dichroism studies.

The interaction between angiotensin II (AII, DRVYIHPF) and its analogs carrying 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid (TOAC) and detergents--negatively charged sodium dodecyl sulfate (SDS) and zwitterionic N-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (HPS)--was examined by means of EPR, CD, and fluorescence. EPR spectra of partially active TOAC1-AII and inactive TOAC3-AII in aqueous solution indicated fast tumbling, the freedom of motion being greater at the N-terminus. Line broadening occurred upon interaction with micelles. Below SDS critical micelle concentration, broader lines indicated complex formation with tighter molecular packing than in micelles. Small changes in hyperfine splittings evinced TOAC location at the micelle-water interface. The interaction with anionic micelles was more effective than with zwitterionic micelles. Peptide-micelle interaction caused fluorescence increase. The TOAC-promoted intramolecular fluorescence quenching was more pronounced for TOAC3-AII because of the proximity between the nitroxide and Tyr4. CD spectra showed that although both AII and TOAC1-AII presented flexible conformations in water, TOAC3-AII displayed conformational restriction because of the TOAC-imposed bend (Schreier et al., Biopolymers 2004, 74, 389). In HPS, conformational changes were observed for the labeled peptides at neutral and basic pH. In SDS, all peptides underwent pH-dependent conformational changes. Although the spectra suggested similar folds for AII and TOAC1-AII, different conformations were acquired by TOAC3-AII. The membrane environment has been hypothesized to shift conformational equilibria so as to stabilize the receptor-bound conformation of ligands. The fact that TOAC3-AII is unable to acquire conformations similar to those of native AII and partially active TOAC1-AII is probably the explanation for its lack of biological activity.

Conformational and functional studies of gomesin analogues by CD, EPR and fluorescence spectroscopies.

The aim of this work was to examine the bioactivity and the conformational behavior of some gomesin (Gm) analogues in different environments that mimic the biological membrane/water interface. Thus, manual peptide synthesis was performed by the solid-phase method, antimicrobial activity was evaluated by a liquid growth inhibition assay, and conformational studies were performed making use of several spectroscopic techniques: CD, fluorescence and EPR. [TOAC(1)]-Gm; [TOAC(1), Ser(2,6,11,15)]-Gm; [Trp(7)]-Gm; [Ser(2,6,11,15), Trp(7)]-Gm; [Trp(9)]-Gm; and [Ser(2,6,11,15), Trp(9)]-Gm were synthesized and tested. The results indicated that incorporation of TOAC or Trp caused no significant reduction of antimicrobial activity; the cyclic analogues presented a beta-hairpin conformation similar to that of Gm. All analogues interacted with negatively charged SDS both above and below the detergent's critical micellar concentration (cmc). In contrast, while Gm and [TOAC(1)]-Gm required higher LPC concentrations to bind to micelles of this zwitterionic detergent, the cyclic Trp derivatives and the linear derivatives did not seem to interact with this membrane-mimetic system. These data corroborate previous results that suggest that electrostatic interactions with the lipid bilayer of microorganisms play an important role in the mechanism of action of gomesin. Moreover, the results show that hydrophobic interactions also contribute to membrane binding of this antimicrobial peptide.

Peptide-lipid interaction monitored by spin labeled biologically active melanocortin peptides.

The present work comparatively analyzes the interaction of alpha-MSH and its more potent and long-acting analog [Nle4, D-Phe7]alpha-MSH (NDP-MSH) with lipid bilayers. The peptides were spin labeled with Toac (2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid) at the N-terminal, as those derivatives had been previously shown to keep their full biological activity. Due to the special rigidity of the Toac covalent binding to the peptide molecule, this spin label is highly sensitive to the peptide backbone conformation and dynamics. The peptides were investigated both by the electron spin resonance (ESR) of Toac0 and the time resolved fluorescence of Trp9 present in the peptides. The Toac0 ESR of the membrane-bound peptides indicates that the two peptides are inserted into the bilayer, close to the bilayer surface, in rather similar environments. A residue titration around pKa 7.5, possibly that of His6, can be clearly monitored by peptide-lipid partition. Trp9 time resolved fluorescence indicates that the peptides, and their Toac-labeled derivatives, present rather similar conformations when membrane bound, though Trp9 in NDP-MSH, and in its Toac-labeled derivative, goes somewhat further down into the bilayer. Yet, Toac0 ESR signal shows that the Toac-labeled N-terminal of NDP-MSH is in a shallower position in the bilayer, as compared to the hormone.

Acid-base titration of melanocortin peptides: evidence of Trp rotational conformers interconversion.

Tryptophantime-resolved fluorescence was used to monitor acid-base titration properties of alpha-melanocyte stimulating hormone (alpha-MSH) and the biologically more potent analog [Nle4, D-Phe7]alpha -MSH (NDP-MSH), labeled or not with the paramagnetic amino acid probe 2,2,6,6-tetramthylpiperidine-N-oxyl-4-amino-4-carboxylic acid (Toac). Global analysis of fluorescence decay profiles measured in the pH range between 2.0 and 11.0 showed that, for each peptide, the data could be well fitted to three lifetimes whose values remained constant. The less populated short lifetime component changed little with pH and was ascribed to Trp g+ chi1 rotamer, in which electron transfer deactivation predominates over fluorescence. The long and intermediate lifetime preexponential factors interconverted along that pH interval and the result was interpreted as due to interconversion between Trp g- and trans chi1 rotamers, driven by conformational changes promoted by modifications in the ionization state of side-chain residues. The differences in the extent of interconversion in alpha-MSH and NDP-MSH are indicative of structural differences between the peptides, while titration curves suggest structural similarities between each peptide and its Toac-labeled species, in aqueous solution. Though less sensitive than fluorescence, the Toac electron spin resonance (ESR) isotropic hyperfine splitting parameter can also monitor the titration of side-chain residues located relatively far from the probe.