Conformational basis for the biological activity of TOAC‐labeled angiotensin II and bradykinin: Electron paramagnetic resonance, circular dichroism, and fluorescence studies

Abstract

N-Terminally and internally labeled analogues of the hormones angiotensin (AII, DRVYIHPF) and bradykinin (BK, RPPGFSPFR) were synthesized containing the paramagnetic amino acid 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid (TOAC). TOAC replaced Asp1 (TOAC1-AII) and Val3 (TOAC3-AII) in AII and was inserted prior to Arg1 (TOAC0-BK) and replacing Pro3 (TOAC3-BK) in BK. The peptide conformational properties were examined as a function of trifluoroethanol (TFE) content and pH. Electron paramagnetic resonance spectra were sensitive to both variables and showed that internally labeled analogues yielded rotational correlation times (tauC) considerably larger than N-terminally labeled ones, evincing the greater freedom of motion of the N-terminus. In TFE, tauC increased due to viscosity effects. Calculation of tau(Cpeptide)/tau(CTOAC) ratios indicated that the peptides acquired more folded conformations. Circular dichroism spectra showed that, except for TOAC1-AII in TFE, the N-terminally labeled analogues displayed a conformational behavior similar to that of the parent peptides. In contrast, under all conditions, the TOAC3 derivatives acquired more restricted conformations. Fluorescence spectra of AII and its derivatives were especially sensitive to the ionization of Tyr4. Fluorescence quenching by the nitroxide moiety was much more pronounced for TOAC3-AII. The conformational behavior of the TOAC derivatives bears excellent correlation with their biological activity, since, while the N-terminally labeled peptides were partially active, their internally labeled counterparts were inactive [Nakaie, C. R., et al., Peptides 2002, 23, 65-70]. The data demonstrate that insertion of TOAC in the middle of the peptide chain induces conformational restrictions that lead to loss of backbone flexibility, not allowing the peptides to acquire their receptor-bound conformation.

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

“…Studies of AII conformation in solvents such as acetonitrile, dimethyl sulfoxide (DMSO), and trifluoroethanol (TFE) have been reported by various authors. [8][9][10]16,20,21,23,[26][27][28][29] A smaller number of reports concerns its interaction, and resulting conformation, with model membranes, such as zwitterionic and anionic micelles 27,30,33,34 and phospholipid bilayers. 20,31,32,34,35 These environments have been found to stabilize conformations involving turn formation.…”

“…Although receptor-ligand specific interactions probably play a role in the final receptor-bound conformation, 28 it has been hypothesized that one of the peptide solution conformations could correspond to that of the energetically favorable receptor-bound state and that binding to the lipid bilayer could stabilize this conformation. [36][37][38][39] The lipid bilayer binding step would also play a role in concentrating the ligand and mediating its access to the membrane-bound receptor.…”

“…The peptide is characterized by a high degree of flexibility and several structural models have been suggested based on theoretical calculations [3][4][5][6][7] and on different experimental approaches for the peptide in solution -in aqueous media and in the presence of secondary structure-inducing solvents [8][9][10]16,21,23,[26][27][28][29] and on binding to model membranes. 20,27,[30][31][32][33][34][35] Spectroscopic studies and theoretical calculations have led to the proposal of different conformations for AII in aqueous solution.…”

“…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.…”

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

“…Following with previous studies initiated with AngII and BK, 11,12) these two peptides were now examined under complementary approaches by coupling in their structures, the amino acid-type probe Toac, introduced earlier in the chemistry of peptides. 18,19) Thus, Toac 1 -AngII, Toac 0 -BK and Toac 3 -BK paramagnetically labeled derivatives, earlier evaluated in terms of structure-function relationships, [20][21][22] were tested towards their molecular stability under strong electromagnetic radiation procedure. The reason for this option lied on the fact that as Toac is a stable free radical, it would be affected by the submission approach to strong gamma ray irradiation.…”