X-ray crystallography and nuclear magnetic resonance measurements provide us with atomically resolved structures of an ever-growing number of biomolecules. These static structural snapshots are important to our understanding of biomolecular function, but real biomolecules are dynamic entities that often exploit conformational changes and transient molecular interactions to perform their tasks. Nuclear magnetic resonance methods can follow such structural changes, but only on millisecond timescales under non-equilibrium conditions. Time-resolved X-ray crystallography has recently been used to monitor the photodissociation of CO from myoglobin on a subnanosecond timescale, yet remains challenging to apply more widely. In contrast, two-dimensional infrared spectroscopy, which maps vibrational coupling between molecular groups and hence their relative positions and orientations, is now routinely used to study equilibrium processes on picosecond timescales. Here we show that the extension of this method into the non-equilibrium regime allows us to observe in real time in a short peptide the weakening of an intramolecular hydrogen bond and concomitant opening of a beta-turn. We find that the rate of this process is two orders of magnitude faster than the 'folding speed limit' established for contact formation between protein side chains.

Photo-triggered ␣-helix formation of a 16-residue peptide featuring a built-in conformational photoswitch is monitored by timeresolved IR spectroscopy. An experimental approach with 2-ps time resolution and a scanning range up to 30 s 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.␣-helix folding ͉ femtosecond IR spectroscopy ͉ protein folding C onformational dynamics of peptides and proteins range from subpicosecond fluctuations of backbone dihedral angles (1) to collective motions of large regions of the molecule, extending to milliseconds and longer (2). Attempts to model dynamics of peptides and proteins often adopt a hierarchical view, which implies a separation of time scales generating classes of events that can be treated separately. The lower, faster hierarchical levels are typically handled in a collective statistical fashion, leading to a transitionstate-theory-like picture (3). This picture is justified if the coupling of the processes, occurring on different time and length scales, allows the selection of a reaction coordinate with a well defined barrier for a simplified description (3, 4). However, the overlap of time scales often brings about questions of the applicability of hierarchical models and leads to controversies, such as those about the interplay between hydrophobic collapse, secondary structure formation, and tertiary structure formation in protein folding (5,6). In this article, we report on stretched kinetics, overlapping dynamics of different spectroscopic probes, and oscillations of the transient absorption during ␣-helix formation. These results indicate that even such simple phenomena as the formation of a single stretch of secondary structure are governed by multiple processes, and a separation of their time scales is not given.Except in a few cases (7-9), the kinetics of helix folding has been inferred indirectly from thermal unfolding experiments (10-17) that require a number of assumptions. If one aims to enter a regime in which these approximations are likely to break down, it is clearly preferable to start from a largely unfolded ensemble and observe the relaxation into a helical state. Helix formation has been achieved previously by temperature (T)-jumping a cold denatured peptide (7). Also, photoexcitation of a ruthenium complex attached to a peptide chain has been used as a trigger (8). Although the complex is electronically excited, its large dipole moment promotes helix ...

Ultrafast IR spectroscopy is used to monitor the nonequilibrium backbone dynamics of a cyclic peptide in the amide I vibrational range with picosecond time resolution. A conformational change is induced by means of a photoswitch integrated into the peptide backbone. Although the main conformational change of the backbone is completed after only 20 ps, the subsequent equilibration in the new region of conformational space continues for times >16 ns. Relaxation and equilibration processes of the peptide backbone occur on a discrete hierarchy of time scales. Albeit possessing only a few conformational degrees of freedom compared with a protein, the peptide behaves highly nontrivially and provides insights into the complexity of fast protein folding.

The technique of transient two-dimensional infrared (T2D-IR) spectroscopy is introduced, which extends the advantage of 2D-IR spectroscopy to the investigation of a transient species with picosecond time resolution. The conformational change of a small cyclic peptide is studied in the amide-I spectral range, which is induced by means of a photoswitch integrated into the peptide backbone. Substantial changes are found in the transient 2D-IR spectra at times when the transient 1D spectra show only a minor time dependence, illustrating the information gain accessible from 2D-IR spectroscopy. In contrast to 1D spectroscopy, 2D-IR can distinguish between homogeneous and inhomogeneous broadening. The homogeneous contribution to the total width of the amide-I band changes during the course of the conformational transition, a result that is interpreted in terms of the manner in which the peptide samples its conformational space.