Acid–base titration of melanocortin peptides: Evidence of Trp rotational conformers interconversion

Abstract

Tryptophantime-resolved fluorescence was used to monitor acid-base titration properties of alpha-melanocyte stimulating hormone (alpha-MSH) and the biologically more potent analog [Nle4, D-Phe7]alpha -MSH (NDP-MSH), labeled or not with the paramagnetic amino acid probe 2,2,6,6-tetramthylpiperidine-N-oxyl-4-amino-4-carboxylic acid (Toac). Global analysis of fluorescence decay profiles measured in the pH range between 2.0 and 11.0 showed that, for each peptide, the data could be well fitted to three lifetimes whose values remained constant. The less populated short lifetime component changed little with pH and was ascribed to Trp g+ chi1 rotamer, in which electron transfer deactivation predominates over fluorescence. The long and intermediate lifetime preexponential factors interconverted along that pH interval and the result was interpreted as due to interconversion between Trp g- and trans chi1 rotamers, driven by conformational changes promoted by modifications in the ionization state of side-chain residues. The differences in the extent of interconversion in alpha-MSH and NDP-MSH are indicative of structural differences between the peptides, while titration curves suggest structural similarities between each peptide and its Toac-labeled species, in aqueous solution. Though less sensitive than fluorescence, the Toac electron spin resonance (ESR) isotropic hyperfine splitting parameter can also monitor the titration of side-chain residues located relatively far from the probe.

Biophysical studies suggest a new structural arrangement of crotoxin and provide insights into its toxic mechanism

“…6, panel C). It has been proposed that the multi-exponential decay of Trp in peptides and proteins results from the occurrence of different rotamers and that the pre-exponential factors are related to the relative rotamer populations5051. Our observation of changes in pre-exponential factors due to CTX heterodimer formation is then related to the conformational changes affecting the Trp residues.…”

The spin label amino acid TOAC and its uses in studies of peptides: chemical, physicochemical, spectroscopic, and conformational aspects

“…EPR and time-resolved fluorescence were also used to investigate the interaction between the TOAC-labeled peptides and DMPG (Fernandez et al 2005a). EPR spectra showed that both peptides displayed similar, strongly restricted motion, indicating insertion into the membrane, both in the gel and in the liquid crystalline phases.…”

How Do Rotameric Conformations Influence the Time-Resolved Fluorescence of Tryptophan in Proteins? A Perspective Based on Molecular Modeling and Quantum Chemistry

“…A 10 ns QMD simulation on -melanotropin, using the CHARM M22 all atom force field and a potential of mean force modeling the solvation effects, yielded three 1 rotamers with the gauche + being less populated than the trans and gauchestates [120]. The presence of rotamers was corroborated by tri-exponential fluorescence decay [121,122] and NMR spectra [123]. However, the NMR studies suggested that the Trp9 rotamer populations are predominantly in the trans and gauche -states with the gauche + states being virtually unpopulated.…”

“…A deviation from the Boltzmann distribution is expected to increase with molecule size. Interestingly, while the short fluorescence lifetime component, ascribed to the gauche + state, changed little with medium pH, the amplitudes of the intermediate and long lifetime components, assigned to the trans and gauche -states, depended strongly on medium pH between pH 2 and pH 11, indicating rotamer interconversion [121].…”