Characterization of a conformationally sensitive TOAC spin-labeled substance P

Shafer, Aaron; Nakaie, Clóvis R.; Deupí, Xavier; Bennett, Vicki; Voss, John C.

doi:10.1016/j.peptides.2008.08.002

Cited by 10 publications

(14 citation statements)

References 51 publications

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“…36,51) The Toac-attaching strategy in its sequence has allowed the synthesis of SP analogues with different ability for binding at the cell membrane level. 37) This relevant peptide showed the lowest D 1/2 value (0.5 kGy) amongst the peptides studied in the present work (Fig. 2, Table 1).…”

Section: Sp (Rpkpqqffglm-amide)mentioning

confidence: 59%

“…Structural evaluation will comprise experiments with different spectroscopy such as dichroism circular (CD), fluorescence, etc., and the biological methodologies to be applied will be those already reported in the corresponding published works. 37,47) In the case of the Pro 4 -BK derivative, muscle contractile experiments with stomach fundus of mice was already initiated and its dose-response data involving the pD 2 and E max values (see Materials and Methods) were determined for comparison with the native agonist BK. No evidence for partial or even residual biological function was detected for this derivative.…”

Section: )mentioning

confidence: 99%

“…cally active molecules such as the α-melanocyte-stimulating hormone (α-MSH) (Ac-SYSMEHFRWGKPV-amide) 33,34) and substance P (SP) (RPKPQQFFGLM-amide) [35][36][37] were also investigated. In this two cases and also of the Pro 4 -BK analogue, after the gamma irradiation step, the main generated by-product derivatives were purified by semi-preparative HPLC and further characterized through different experimental techniques including analytical HPLC, LC/electrospray ionization (ESI)-MS, amino acid analysis and mainly by daughter ion scanning by collision induced dissociation (CID-MS/MS) sequencing procedure.…”

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confidence: 99%

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Peptide Structure Modifications: Effect of Radical Species Generated by Controlled Gamma Ray Irradiation Approach

Vieira

Nardi

Nascimento

et al. 2013

Biological & Pharmaceutical Bulletin

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The present work aimed at evaluating the radiolysis effect upon a set of peptides, most of them involved in physiological functions. To generate reactive radical species, a Co 60 source (up to 15 kGy) was used for controlled gamma irradiation of some peptide solutions including derivatives attaching the stable free radical Toac (2,2,6,6-tetramethypiperidine-1-oxyl-4-amino-4-carboxylic acid). Regardless of the peptide sequence, a nonlinear and progressive degradation of a total of nine peptides was detected. The results were interpreted in the light of the half-life dose (D 1/2 ) parameter which represents the dose necessary for 50% peptide structure degradation. The vasoactive angiotensin II (AngII)'s analogue Ang-(1-7) showed greater stability towards gamma ray radiation than bradykinin (BK), Toac 0 -BK, Pro 4 -BK (D 1/2 around 4 and 2 kGy, respectively) which decreased to about 0.5-1.0 kGy in the case of acetyl-α-melanocyte-stimulating hormone (Ac-α-MSH) and substance P (SP). In terms of peptide structural modifications, the data acquired from different analytical methods suggested a Phe to Tyr (or its ortho and/or meta isomers) transformation as a consequence of the hydroxyl moiety insertion. Noteworthy, this effect seemed to be position-dependent as only Phe located at or near the C-terminal portion seemed to display this transformation. In contrast, Met is comparatively more easily oxidized, thus allowing to conclude that gamma irradiation may induce a complex position and/ or sequence-dependent effect on peptides. As previously applied for BK, some irradiated peptides were submitted to their by-products purification, indeed a complementary target of the present approach for development of uncommon analogues for further structure-function investigation.

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Section: Sp (Rpkpqqffglm-amide)mentioning

confidence: 59%

Section: )mentioning

confidence: 99%

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confidence: 99%

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Peptide Structure Modifications: Effect of Radical Species Generated by Controlled Gamma Ray Irradiation Approach

Vieira

Nardi

Nascimento

et al. 2013

Biological & Pharmaceutical Bulletin

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“…R. Balog et al, 2004; Kalai, Schindler, Balog, Fogassy, & Hideg, 2008; Tominaga et al, 2001; Wright et al, 2007). The most commonly used one is 2,2,6,6-tetramethyl-N-oxyl-4-amino-4-carboxylic acid (TOAC, Figure 1, 8 ) (Rassat & Rey, 1967; Schreier, Bozelli, Marín, Vieira, & Nakaie, 2012; A. M. Shafer, Nakaie, Deupi, Bennett, & Voss, 2008; Thomas et al, 2005).…”

Section: Peptide Labeling With Epr Active Probes and Preparation Omentioning

confidence: 99%

Peptide–Membrane Interactions by Spin-Labeling EPR

Smirnova

Smirnov

2015

Methods in Enzymology

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Site-directed spin labeling (SDSL) in combination with Electron Paramagnetic Resonance (EPR) spectroscopy is a well-established method that has recently grown in popularity as an experimental technique, with multiple applications in protein and peptide science. The growth is driven by development of labeling strategies, as well as by considerable technical advances in the field, that are paralleled by an increased availability of EPR instrumentation. While the method requires an introduction of a paramagnetic probe at a well-defined position in a peptide sequence, it has been shown to be minimally destructive to the peptide structure and energetics of the peptide-membrane interactions. In this chapter, we describe basic approaches for using SDSL EPR spectroscopy to study interactions between small peptides and biological membranes or membrane mimetic systems. We focus on experimental approaches to quantify peptide-membrane binding, topology of bound peptides, and characterize peptide aggregation. Sample preparation protocols including spin-labeling methods and preparation of membrane mimetic systems are also described.

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“…43 The probe's C a -tetrasubstituted cyclic structure favors acquisition of helical structure, as well as turn formation 44,45 and renders the EPR spectra of labeled (macro)molecules highly sensitive to the backbone conformational properties. Owing to these unique characteristics, a large number of studies focusing on peptides, including our own 28,[46][47][48][49][50][51][52][53][54][55][56][57][58] and from a variety of groups [59][60][61][62][63][64][65][66][67][68][69][70][71][72][73][74] appeared in the literature in recent years. In addition to its applications in structural studies of peptides, TOAC has also been used advantageously in solid phase peptide synthesis to monitor peptide chain assembly throughout the polymer backbone.…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2009

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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.

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Characterization of a conformationally sensitive TOAC spin-labeled substance P

Cited by 10 publications

References 51 publications

Peptide Structure Modifications: Effect of Radical Species Generated by Controlled Gamma Ray Irradiation Approach

Peptide Structure Modifications: Effect of Radical Species Generated by Controlled Gamma Ray Irradiation Approach

Peptide–Membrane Interactions by Spin-Labeling EPR

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

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