Conformational dynamics of peptides: a fluorescence study.

Author(s)

Eduardo Sérgio de Souza

• Document type: Doctoral Thesis
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Instituto de Física (IF/SBI)
• Defense date: 2004-11-05

Examining board members

Amando Siuiti Ito; Iouri Borissevitch; Sylvio Roberto Accioly Canuto; Vera Bohomoletz Henriques; Hidetake Imasato

• Advisor: Amando Siuiti Ito

Abstract

Steady state and time-resolved fluorescence spectroscopy were used to study the conformational dynamics of three families of peptides: the carboxyl terminal of the neuroendocrine peptide (782Cl), the alpha melanocytestimulating hormone (alpha-MSH) and heparin-binding consensus sequence peptides (Cardin motif peptides). The fluorescence methods were based on quenching of the emission of a fluorophore present in the peptide, either by collisional interaction with a quencher, as by energy transfer to an acceptor group. In the second case, within the hypothesis that different peptide conformations are in equilibrium during the fluorescence decay, we supposed that the intensity decay was modulated by an acceptor-donor distance distribution function, f(r). A computational procedure was developed using the CONTIN program, to recover, from the time-resolved fluorescence data, the distance distribution between donor and acceptor groups attached to the peptides. The methodology proved to be useful to provide quantitative information about conformational dynamics of peptides, its dependency on the solvent and its interaction with other molecules present in the medium. The peptide 782CT and its analogs A12 and A18 were studied by examination of fluorescence from the tyrosine residue present in the position five of the amino acid sequence. There were no appreciable differences in the intensity decay, anisotropy decay and iodide quenching between the three peptides, that could be correlated to observed differences in the inhibition of the pro-hormane convertase 2 (PC2). The peptide hormone alpha-MSH and its more potent analog [Nle(4),0- Phe(7)]alpha-MSH (NDP-alpha-MSH), containing the fluorescent residue tryptophan, were labeled at the amino terminal with the amino benzoic acid (Abz) group, and were also examined by fluorescence methods. We observed energy transfer between the tryptophan residue acting as donor and Abz as acceptor, and verified a broad Trp-o-Abz distance distribution for alpha-MSH in aqueous medium, while three different distance populations could be identified for the labeled analog NDP-alpha-MSH in water, indicating distinct conformational states for the synthetic peptide, compared with the native hormone. Measurements in trifluoroethanol resulted in the recovery of two Abzlrp distance populations, both for the native and the analog hormones, reflecting the decrease, induced by the solvent, of the conformational states available to the peptides. The Cardin motif peptides were examined in the presence of heparin, SDS micelles and in TFE/water mixtures. The fluorescent group Abz was attached to the amino terminal and the acceptor group N-(2,4 dinitrophenyl)ethylenediamine (Eddnp) was bound to the carboxyl terminal of the peptides. The end-to-end distance distribution recovered from time-resolved fluorescence data indicated extend conformation in aqueous medium and the occurrence of compact conformations under interaction with heparin and SDS or in the medium containing TFE. The end-to-end distances recovered were lower than those expected for the peptides in alpha helix conformation, suggesting the occurrence of turned structures beyond the alpha helix. The energy transfer methodology shows to be useful to study conformational dynamics of peptides in solution. (AU)