Peptide Folding Dynamics:  A Time-Resolved Study from the Nanosecond to the Microsecond Time Regime

Abstract

Time-resolved spectroscopies, spanning from the nanosecond to the microsecond time regime, coupled with molecular mechanics calculations, allowed us to assess the most populated conformations in solution of a series of analogues of trichogin GA IV, a natural undecapeptide showing significant antimicrobial activity. This peptide is characterized by a high content of the conformationally constrained alpha-aminoisobutyric acid and by a glycine-glycine motif in the central part of the sequence. Nanosecond time-resolved fluorescence experiments were performed to determine the conformational properties of the peptide analogues in solution, while transient absorption measurements allowed us to study the peptide dynamics on the microsecond time scale. Because the peptides examined were functionalized by a fluorescent probe at the N-terminus and a nitroxide quencher placed along the backbone at three different positions, the distance-dependent fluorophore-quencher interaction was exploited to obtain a deeper insight into their three-dimensional structural and dynamical properties. Further information on the conformational and dynamical features was obtained by photophysical experiments as a function of the viscosity and polarity of the medium. Taken together, the results revealed a transition from an elongated, helical conformation to a family of compact, folded structures mimicking a helix-turn-helix motif, which may represent a model of the early steps of the protein hydrophobic collapse.

Metal Binding Properties of Fluorescent Analogues of Trichogin GA IV: A Conformational Study by Time‐Resolved Spectroscopy and Molecular Mechanics Investigations

“…It has already been reported that while substitution of the Fmoc group does not affect the conformational properties of the trichogin analogues, inclusion of TOAC in the C-terminal segment slightly stabilizes the helical conformation. [15] Permeabilization experiments on phospholipid bilayers proved that the above substitutions do not perturb the membrane activity of the peptides investigated. [14] The metal ion binding properties of two fluorescent analogues of trichogin GA IV, which is a natural undecapeptide showing significant antimicrobial activity, were studied by circular dichroism, time-resolved optical spectroscopy, and molecular mechanics calculations.…”

“…[14,15] It has been shown that for Fmoc···TOAC centre-tocentre distances longer than 4-5 , the Fçrster dipole-dipole interaction model [27] adequately describes the excited state interaction between the aromatic molecule and the nitroxyl radical quencher. [28] At shorter distances, the overlap of the electronic distributions gives rise to ultrafast relaxation mechanisms that completely deplete the fluorescence emission.…”

“…[11][12][13] Recently, we investigated the conformational and dynamic properties in solution of fluorescent labelled TrGA analogues by timeresolved optical spectroscopies and molecular mechanics (MM) calculations, and showed that a conformational transition from an elongated, helical conformation to a family of compact, folded structures that mimic a helix-turn-helix motif, takes place in the microsecond time regime. [14,15] The close similarity of these folded conformers to the helixloop-helix motif of a number of calcium-binding proteins and peptides [16,17] urged us to investigate the metal-ion binding properties of these TrGA analogues. Besides Ca II , we selected Gd III for its well-known properties as a magnetic probe and because due to its similar radius and coordination properties is the best substitute for calcium.…”

“…[14,15] Information on the peptide dynamics in the microsecond time region was also obtained from time-resolved transient absorption measurements by analysing the quenching of the Fmoc triplet state decay by TOAC. Summarising the principal findings of our previous investigations, we showed that the F0T8 dynamics in the microsecond time region is determined by a conformational transition from an extended helical conformation, similar to the 3 10 /a-helix mixed conformation revealed by X-ray diffraction and NMR spectroscopy studies, to a family of compact, folded structures characterized by a b bend involving the Aib4-Gly5-Gly6 motif.…”

“…Summarising the principal findings of our previous investigations, we showed that the F0T8 dynamics in the microsecond time region is determined by a conformational transition from an extended helical conformation, similar to the 3 10 /a-helix mixed conformation revealed by X-ray diffraction and NMR spectroscopy studies, to a family of compact, folded structures characterized by a b bend involving the Aib4-Gly5-Gly6 motif. [15] This transition requires an activated torsional motion, that is, a + 1208 rotation around the Gly6 f torsional angle, which is characterized by a rate constant of 3.1 10 6 s À1 (320 ns). [7] This approach was applied here for investigating the conformational and dynamic properties of the ion-peptide complexes formed by Ca II and Gd III with the aforementioned TrGA fluorescent analogues.…”