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 (τ_C) considerably larger than N‐terminally labeled ones, evincing the greater freedom of motion of the N‐terminus. In TFE, τ_C increased due to viscosity effects. Calculation of τ_Cpeptide/τ_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. © 2004 Wiley Periodicals, Inc. Biopolymers, 2004