Renata F. F. Vieira

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 β-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.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 β-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.

Comparative EPR and fluorescence conformational studies of fully active spin-labeled melanotropic peptides

Similar to melanocyte stimulating hormone (α‐MSH), its potent and long‐acting analogue, [Nle4, D‐Phe7]α‐MSH, when labeled with the paramagnetic amino acid probe 2,2,6,6‐tetramethylpiperidine‐N‐oxyl‐4‐amino‐4‐carboxylic acid (Toac), maintains its full biological potency, thus validating any comparative structural investigations between the two labeled peptides. Correlation times, calculated from the electron paramagnetic resonance signal of Toac bound to the peptides, and Toac–Trp distances, estimated from the Toac fluorescence quenching of the Trp residue present in the peptides, indicate a more rigid and folded structure for the potent analogue as compared to the hormone, in aqueous medium.

Antiplasmodial activity study of angiotensin II via Ala scan analogs

Angiotensin II (AII) as well as analog peptides shows antimalarial activity against Plasmodium gallinaceum and Plasmodium falciparum, but the exact mechanism of action is still unknown. This work presents the solid‐phase synthesis and characterization of eight peptides corresponding to the alanine scanning series of AII plus the amide‐capped derivative and the evaluation of the antiplasmodial activity of these peptides against mature P. gallinaceum sporozoites. The Ala screening data indicates that the replacement of either the Ile5 or the His6 residues causes minor effects on the in vitro antiplasmodial activity compared with AII, i.e. AII (88%), [Ala6]‐AII (79%), and [Ala5]‐AII (75%). Analogs [Ala3]‐AII, [Ala1]‐AII, and AII‐NH2 showed antiplasmodial activity around 65%, whereas the activity of the [Ala8]‐AII, [Ala7]‐AII, [Ala4]‐AII, and [Ala2]‐AII analogs is lower than 45%. Circular dichroism data suggest that AII and the most active analogs adopt a β‐fold conformation in different solutions. All AII analogs, except [Ala4]‐AII and [Ala8]‐AII, show contractile responses and interact with the AT1 receptor, [Ala5]‐AII and [Ala6]‐AII. In conclusion, this approach is helpful to understand the contribution of each amino acid residue to the bioactivity of AII, opening new perspectives toward the design of new sporozoiticidal compounds. Copyright

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.

A Proposed EPR Approach to Evaluating Agonist Binding Site of a Peptide Receptor

Angiotensin II (Ang II) and its transmembrane AT1 receptor were selected in order to test an innovative strategy that might allow the assessment of the agonist binding site in the receptor molecule. With the use of the 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid (TOAC) paramagnetic probe, a biologically active agonist (TOAC1-Ang II), as well as an inactive control (TOAC4-Ang II) analogs were mixed in solution with various synthesized AT1 fragments. Comparative intermolecular interactions, as estimated by analyzing the EPR spectra of solutions, suggested the existence of an agonist binding site containing a sequence composed of portions of the N-terminal (13-17) and the third extracellular loop (266-278) fragments of the AT1 molecule. Therefore, this combined EPR-TOAC approach shows promise as an alternative for use also in other applications related to specific intermolecular association processes.

Acid–base titration of melanocortin peptides: Evidence of Trp rotational conformers interconversion

Tryptophantime‐resolved fluorescence was used to monitor acid–base titration properties of α‐melanocyte stimulating hormone (α‐MSH) and the biologically more potent analog [Nle4, D‐Phe7]α ‐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+ χ1 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 χ1 rotamers, driven by conformational changes promoted by modifications in the ionization state of side‐chain residues. The differences in the extent of interconversion in α‐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.

Conformational studies of TOAC-labeled bradykinin analogues in model membranes

Spin-labeled analogues of bradykinin (BK) were synthesized containing the amino acid TOAC (2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid) either before Arg1 (TOAC0-BK) or replacing Pro3 (TOAC3-BK). Whereas the latter is inactive, the former retains about 70% of BKs activity in isolated rat uterus. A combined electron paramagnetic resonance (EPR)-circular dichroism (CD) approach was used to examine the conformational properties of the peptides in the presence of membrane-mimetic systems (negatively charged sodium dodecyl sulfate, SDS, and zwitterionic N-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, HPS). While the peptides bind to both monomeric and micellar SDS, no interaction occurs with HPS, evincing the contribution of electrostatic interactions. TOAC3-BKs EPR spectral lineshapes are broader than those of TOAC0-BK, indicating a more restricted degree of motion at position 3. Moreover, the motional freedom of both peptides decreased upon binding to SDS. BK and TOAC0-BK solution CD spectra indicate highly flexible conformations (possibly an equilibrium between rapidly interconverting forms), while TOAC3-BKs spectra correspond to a more ordered structure. SDS binding induces drastic changes in BK and TOAC0-BK spectra, indicating stabilization of similar folds. The micelle interface promotes a higher degree of secondary structure by favoring intramolecular hydrogen bonds. In contrast. TOAC3-BK spectra remain essentially unchanged. These results are interpreted as due to TOACs ring imposing a more constrained conformation. This rigidity is very likely responsible for the inability of TOAC3-BK to acquire the correct receptor-bound conformation leading to loss of biological activity, On the other hand, the greater flexibility of TOAC0-BK and the similarity between its conformational behavior and that of the native hormone are probably related to their similar biological activity.

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.

Short peptide constructs mimic agonist sites of AT1R 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 AT1R and BKRB1 or BKRB2 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 (Toac1-AngII and Toac0-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 Toac3-AngII and Toac3-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 AT1R and BKRB cyclic constructs based on the structure of the CXCR4, an α-chemokine GPCR-type receptor.

Alternative and simple normal-phase HPLC enantioseparation of a chiral amino acid-type spin label derivative

Neste trabalho desenvolveu-se um processo alternativo para separacao cromatografica dos enantiomeros (+)-(3R,4R) e ( - )-(3S,4S) do β-aminoacido quiral trans-2,2,5,5-tetrametilpirrolidina-3-amino-4-carboxilico (POAC), que estava protegido no grupo aminico para posterior ligacao a um peptideo, polimero ou outra macromolecula. A enantioseparacao foi obtida por HPLC usando uma fase estacionaria normal a base de celulose quiral e eluicao isocratica. O sistema n-hexano:isopropanol, sempre com maior quantidade do primeiro solvente, foi usado como fase movel, pois forneceu os melhores resultados na separacao dos dois componentes, constatado pelos valores mais elevados de fator de separacao e de indice de resolucao cromatografica. Estes parâmetros apresentaram valores de 3,7 e 18,4 e de 2,0 e 6,7 nas solucoes com proporcao 90:10 (v/v) e 80:20 (v/v) de n-hexano:isopropanol, respectivamente. Estes dados indicam que a estrategia de purificacao cromatografica em uma unica etapa usando fase normal e viavel, abrindo assim a perspectiva de uma producao rapida e em grande escala desta sonda paramagnetica.