Conformational Properties of Seven Toac-Labeled Angiotensin I Analogues Correlate with Their Muscle Contraction Activity and Their Ability to Act as ACE Substrates

Abstract

Conformational properties of the angiotensin II precursor, angiotensin I (AngI) and analogues containing the paramagnetic amino acid TOAC (2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid) at positions 0, 1, 3, 5, 8, 9, and 10, were examined by EPR, CD, and fluorescence. The conformational data were correlated to their activity in muscle contraction experiments and to their properties as substrates of the angiotensin I-converting enzyme (ACE). Biological activity studies indicated that TOAC0-AngI and TOAC1-AngI maintained partial potency in guinea pig ileum and rat uterus. Kinetic parameters revealed that only derivatives labeled closer to the N-terminus (positions 0, 1, 3, and 5) were hydrolyzed by ACE, indicating that peptides bearing the TOAC moiety far from the ACE cleavage site (Phe8-His9 peptide bond) were susceptible to hydrolysis, albeit less effectively than the parent compound. CD spectra indicated that AngI exhibited a flexible structure resulting from equilibrium between different conformers. While the conformation of N-terminally-labeled derivatives was similar to that of the native peptide, a greater propensity to acquire folded structures was observed for internally-labeled, as well as C-terminally labeled, analogues. These structures were stabilized in secondary structure-inducing agent, TFE. Different analogues gave rise to different β-turns. EPR spectra in aqueous solution also distinguished between N-terminally, internally-, and C-terminally labeled peptides, yielding narrower lines, indicative of greater mobility for the former. Interestingly, the spectra of peptides labeled at, or close, to the C-terminus, showed that the motion in this part of the peptides was intermediate between that of N-terminally and internally-labeled peptides, in agreement with the suggestion of turn formation provided by the CD spectra. Quenching of the Tyr4 fluorescence by the differently positioned TOAC residues corroborated the data obtained by the other spectroscopic techniques. Lastly, we demonstrated the feasibility of monitoring the progress of ACE-catalyzed hydrolysis of TOAC-labeled peptides by following time-dependent changes in their EPR spectra.

Fig 1. EPR spectra of TOAC⁰-AngI (A), TOAC¹-AngI (B), TOAC³-AngI (C), TOAC⁵-AngI (D), TOAC⁸-AngI (E), TOAC⁹-AngI (F) and TOAC¹⁰-AngI (G) in 20 mM phosphate buffer, pH 7.0.

Field scan: 100 Gauss.

Fig 2. CD spectra of AngI (A), TOAC⁰-AngI (B), TOAC¹-AngI (C) TOAC³-AngI (D), TOAC⁵-AngI (E), TOAC⁸-AngI (F), TOAC⁹-AngI (G) and TOAC¹⁰-AngI (H) as a function of TFE concentration.

Fig 3. Fluorescence spectra of AngI and TOAC-AngI analogues in 20 mM phosphate buffer, pH 7.

Fig 4. Kinetics of ACE-catalyzed hydrolysis of TOAC⁰-AngI (A), TOAC¹-AngI (B), TOAC³-AngI (C), and TOAC⁵-AngI (D) monitored by the variation of the h₀/h_-1 values in the peptides EPR spectra as a function of time.