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

Similar to melanocyte stimulating hormone (alpha -MSH), its potent and long-acting analogue, [Nle(4), D-Phe(7)]alpha -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 tire 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. (C) 2001 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.Universidade Federal de São Paulo, Dept Biofis, BR-04044020 São Paulo, BrazilUniv São Paulo, Inst Fis, BR-66318 São Paulo, BrazilUniv São Paulo, Inst Biociencias, Dept Fisiol, BR-11176 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biofis, BR-04044020 São Paulo, BrazilWeb of Scienc

Ortho-aminobenzoic acid-labeled bradykinins in interaction with lipid vesicles: Fluorescence study

The peptide hormone bradykinin (BK) (Arg(1)-Pro(2)-Pro(3)-Gly(4)-Phe(5)-Ser(6)-Pro(7)-Phe(8)-Arg(9)) and its shorter homolog BK1-5 (Arg(1)-Pro(2)-Pro(3)-Gly(4)-Phe(5)) were labeled with the extrinsic fluorescent probe ortho-aminobenzoic acid (Abz) bound to the N-terminal and amidated in the C-terminal carboxyl group (Abz-BK-NH2 and Abz-BK1-5-NH2). the fragment des-Arg(9)-BK was synthesized with the Abz fluorescent probe attached to the 3-amino group of 2,3-amino propionic acid (DAP), which positioned the Abz group at the C-terminal side of BK sequence, constituting the peptide des-Arg(9)-BK-DAP(Abz)-NH2. the spectral characteristics of the probe were similar in the three peptides, and their fluorescent properties were monitored to study the interaction of the peptides with anionic vesicles of dimyristoylphosphatidylglycerol (DMPG). Time-resolved fluorescence experiments showed that the fluorescence decay of the peptides was best described by double-exponential kinetics, with mean lifetimes values around 8.0 ns in buffer pH 7.4 that increased about 10% in the presence of DMPG vesicles. About a 10-fold increase, compared with the values in aqueous solution, was observed in the steady-state anisotropy in the presence of vesicles. A similar increase was also observed for the rotational correlation times obtained from time-resolved anisotropy decay profiles, and related to the overall tumbling of the peptides. Equilibrium binding constants for the peptide-lipid interaction were examined monitoring anisotropy values in titration experiments and the electrostatic effects were evaluated through Gouy-Chapman potential calculations. Without corrections for electrostatic effects, the labeled fragment Abz-BK1-5-NH2 presented the major affinity for DMPG vesicles. Corrections for the changes in peptide concentration due to electrostatic interactions suggested higher affinity of the BK fragments to the hydrophobic phase of the bilayer. (C) 2002 Wiley Periodicals, Inc.Univ São Paulo, Fac Filosofia Ciencias & Letras Ribeirao Pret, Dept Fis & Matemat, BR-14040901 Ribeirao Preto, BrazilUniv São Paulo, Inst Fis, BR-14040901 Ribeirao Preto, BrazilUniversidade Federal de São Paulo, Dept Biofis, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biofis, São Paulo, BrazilWeb of Scienc