Alpha-helix formation in a photoswitchable peptide tracked from picoseconds to microseconds by time-resolved IR spectroscopy

Abstract

Photo-triggered alpha-helix formation of a 16-residue peptide featuring a built-in conformational photoswitch is monitored by time-resolved IR spectroscopy. An experimental approach with 2-ps time resolution and a scanning range up to 30 micros is used to cover all time scales of the peptide dynamics. Experiments are carried out at different temperatures between 281 and 322 K. We observe single-exponential kinetics of the amide I' band at 322 K on a time scale comparable to a recent temperature-jump folding experiment. When lowering the temperature, the kinetics become slower and nonexponential. The transition is strongly activated. Spectrally dispersed IR measurements provide multiple spectroscopic probes simultaneously in one experiment by resolving the amide I' band, isotope-labeled amino acid residues, and side chains. We find differing relaxation dynamics at different spectral positions.

Fig. 1.

The photoswitchable FK11X peptide. (A) Cross-linker in cis (Left) and trans (Right) conformation. (B) FK11X sequence, showing the positions of the ¹³C labels (underlined residues) and the attached cross-linker. (C) Schematic models of cis-FK11X (Left) and trans-FK11X (Right), illustrating the conformational transition induced by the photoswitchable linker. Hydrogens and side chains are omitted for clarity.

Fig. 2.

IR spectra. (Upper) Fourier transform IR (FTIR) absorption spectra of trans-FK11X(iso) and FK11X. (Lower) Transient difference spectra of FK11X(iso) at different delays and magic angle polarization. Steady-state Fourier transform IR difference spectra of FK11X(iso) and FK11X. All measurements were taken at 20°C. See Experimental Results and Assignments for discussion of numbered peaks.

Fig. 3.

Time-dependent amplitude of the amide I′ difference signal at different temperatures and exponential and stretched exponential fits. (Inset) Dots, Arrhenius plot of the rates (taken from exponential fits); solid line, fit using Eq. 2 assuming non-Arrhenius (quadratic in 1/T, where T is temperature) behavior. Within the small temperature interval we cannot distinguish an Arrhenius (linear in 1/T) from a quadratic dependence on 1/T.

Fig. 4.

Dynamics of selected spectral positions in the early nanosecond range at 20°C. (A) Decreasing absorption at the isotope position. (B) Decrease at the isotope position continues until 100 ns, by which time build up at the amide I′ and Glu position has begun already. Only after 100 ns does the absorption at the isotope position rise. (C) Dynamics at selected spectral positions. The solid lines are fits with stretched exponentials.

Fig. 5.

A simple model explaining the experimental observations. The solid line is a free-energy surface, and the dotted line represents the enthalpic contribution only. The roughness of the unfolded manifolded is characterized by ΔH.