RESUMO
Fibroblast growth factors (FGFs) are regulatory proteins associated with a number of physiological and pathological states. On the basis of data suggesting a functional role for specific regions of human acidic FGF (aFGF), a linear peptide encompassing residues 99-108 (peptide1) and its cyclic analogue (peptide 2) were synthesized and their functional and structural features were investigated. While peptide 1 is inactive on Balb/c 3T3 fibroblasts, peptide 2 is mitogenic with ED(50) of approximately 50 microM. Moreover, peptide 1 is not able to inhibit the binding of human aFGF to cellular receptors whereas peptide 2 exhibits significant inhibitory activity. The NMR-derived solution conformers indicated the presence, only in peptide 2, of structural elements that we believe are related to its ability to emulate the biological activity of the native protein. These results suggest that the expression of mitogenic activity in short peptides, besides the presence of specific amino acids, requires the existence of stable structural features. In addition, they indicate that the introduction of chemical restraints in peptides can provide novel possibilities for the development of receptor agonists or antagonists.
Assuntos
Fator 1 de Crescimento de Fibroblastos/química , Fatores de Crescimento de Fibroblastos/síntese química , Mitógenos/síntese química , Oligopeptídeos/síntese química , Fragmentos de Peptídeos/síntese química , Peptídeos Cíclicos/síntese química , Células 3T3 , Animais , Dicroísmo Circular , Fatores de Crescimento de Fibroblastos/química , Fatores de Crescimento de Fibroblastos/farmacologia , Humanos , Espectroscopia de Ressonância Magnética , Camundongos , Mitógenos/química , Mitógenos/farmacologia , Modelos Moleculares , Oligopeptídeos/química , Oligopeptídeos/farmacologia , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/farmacologia , Peptídeos Cíclicos/química , Peptídeos Cíclicos/farmacologia , Conformação Proteica , Timidina/metabolismoRESUMO
We examined the effect of uncharged lidocaine on the structure and dynamics of egg phosphatidylcholine (EPC) membranes at pH 10.5 in order to assess the location of this local anesthetic in the bilayer. Changes in the organization of small unilamellar vesicles were monitored either by electron paramagnetic resonance (EPR)-in the spectra of doxyl derivatives of stearic acid methyl esters labeled at different positions in the acyl chain (5-, 7-, 12- and 16-MeSL)-or by fluorescence, with pyrene fatty-acid (4-, 6-, 10- and 16-Py) probes. The largest effects were observed with labels located at the upper positions of the fatty-acid acyl-chain. Dynamic information was obtained by 1H-NMR. Lidocaine protons presented shorter longitudinal relaxation times (T(1)) values due to their binding, and consequent immobilization to the membrane. In the presence of lidocaine the mobility of all glycerol protons of EPC decreased, while the choline protons revealed a higher degree of mobility, indicating a reduced participation in lipid-lipid interactions. Two-dimensional Nuclear Overhauser Effect experiments detected contacts between aromatic lidocaine protons and the phospholipid-choline methyl group. Fourier-transform infrared spectroscopy spectra revealed that lidocaine changes the access of water to the glycerol region of the bilayer. A "transient site" model for lidocaine preferential location in EPC bilayers is proposed. The model is based on the consideration that insertion of the bulky aromatic ring of the anesthetic into the glycerol backbone region causes a decrease in the mobility of that EPC region (T(1) data) and an increased mobility of the acyl chains (EPR and fluorescence data).
Assuntos
Lidocaína/química , Bicamadas Lipídicas/química , Fosfatidilcolinas/química , Óxido de Deutério/química , Espectroscopia de Ressonância de Spin Eletrônica , Liofilização , Espectroscopia de Ressonância Magnética , Microscopia de Fluorescência , Modelos Moleculares , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
The conformation of three synthetic peptides encompassing the proximal and distal half of the third intracellular loop (Ni3 and Ci3) and a portion of the cytoplasmic tail (fCT) of the angiotensin II AT1A receptor has been studied using circular dischroism and fluorescence spectroscopies. The results show that the conformation of the peptides is modulated in various ways by the environmental conditions (pH, ionic strength and dielectric constant). Indeed, Ni3 and fCT fold into helical structures that possess distinct stability and polarity due to the diverse forces involved: mainly polar interactions in the first case and a combination of polar and hydrophobic interactions in the second. The presence of these various features also produce distinct intermolecular interactions. Ci3, instead, exists as an ensemble of partially folded states in equilibrium. Since the corresponding regions of the angiotensin II AT1A receptor are known to play an important role in the receptor function, due to their ability to undergo conformational changes, these data provide some new clues about their different conformational plasticity.