RESUMO
Extracellular peptide ligand binding sites, which bind the N-termini of angiotensin II (AngII) and bradykinin (BK) peptides, are located on the N-terminal and extracellular loop 3 regions of the AT(1)R and BKRB(1) or BKRB(2) G-protein-coupled receptors (GPCRs). Here we synthesized peptides P15 and P13 corresponding to these receptor fragments and showed that only constructs in which these peptides were linked by S-S bond, and cyclized by closing the gap between them, could bind agonists. The formation of construct-agonist complexes was revealed by electron paramagnetic resonance spectra and fluorescence measurements of spin labeled biologically active analogs of AngII and BK (Toac(1)-AngII and Toac(0)-BK), where Toac is the amino acid-type paramagnetic and fluorescence quencher 2, 2, 6, 6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid. The inactive derivatives Toac(3)-AngII and Toac(3)-BK were used as controls. The interactions characterized by a significant immobilization of Toac and quenching of fluorescence in complexes between agonists and cyclic constructs were specific for each system of peptide-receptor construct assayed since no crossed reactions or reaction with inactive peptides could be detected. Similarities among AT, BKR, and chemokine receptors were identified, thus resulting in a configuration for AT(1)R and BKRB cyclic constructs based on the structure of the CXCR(4), an α-chemokine GPCR-type receptor.
Assuntos
Angiotensina II/agonistas , Bradicinina/agonistas , Peptídeos/química , Receptor Tipo 1 de Angiotensina/química , Receptores da Bradicinina/química , Sequência de Aminoácidos , Angiotensina II/genética , Angiotensina II/metabolismo , Sítios de Ligação , Bradicinina/genética , Bradicinina/metabolismo , Espectroscopia de Ressonância de Spin Eletrônica , Dados de Sequência Molecular , Peptídeos/genética , Peptídeos/metabolismo , Ligação Proteica , Estrutura Secundária de Proteína , Receptor Tipo 1 de Angiotensina/genética , Receptor Tipo 1 de Angiotensina/metabolismo , Receptores da Bradicinina/genética , Receptores da Bradicinina/metabolismoRESUMO
Based on solvation studies of polymers, the sum (1:1) of the electron acceptor (AN) and electron donor (DN) values of solvents has been proposed as an alternative polarity scale. To test this, the electron paramagnetic resonance isotropic hyperfine splitting constant, a parameter known to be dependent on the polarity/proticity of the medium, was correlated with the (AN+DN) term using three paramagnetic probes. The linear regression coefficient calculated for 15 different solvents was approximately 0.9, quite similar to those of other well-known polarity parameters, attesting to the validity of the (AN+DN) term as a novel "two-parameter" solvent polarity scale.
