Jens Bredenbeck

Picosecond conformational transition and equilibration of a cyclic peptide

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.

Double-resonance versus pulsed Fourier transform two-dimensional infrared spectroscopy: An experimental and theoretical comparison

In this study we focus on the differences and analogies of two experimental implementations of two-dimensional infrared (2D-IR) spectroscopy: double-resonance or dynamic hole burning 2D-IR spectroscopy and pulsed Fourier transform or heterodyne detected photon echo spectroscopy. A comparison is done theoretically as well as experimentally by contrasting data obtained from both methods. As an example we have studied the strongly coupled asymmetric and symmetric carbonyl stretching vibrations of dicarbonylacetylacetonato rhodium dissolved in hexane. Both methods yield the same peaks in a 2D-IR spectrum. Within certain approximations we derive an analytic expression which shows that the 2D-IR spectra are broadened in one frequency dimension in the double-resonance experiment by convolution with the pump pulse spectral width, while the spectral resolution in the other frequency direction is the same in both cases.

α-Helix folding in the presence of structural constraints

We have investigated the site-specific folding kinetics of a photoswitchable cross-linked α-helical peptide by using single 13C = 18O isotope labeling together with time-resolved IR spectroscopy. We observe that the folding times differ from site to site by a factor of eight at low temperatures (6°C), whereas at high temperatures (45°C), the spread is considerably smaller. The trivial sum of the site signals coincides with the overall folding signal of the unlabeled peptide, and different sites fold in a noncooperative manner. Moreover, one of the sites exhibits a decrease of hydrogen bonding upon folding, implying that the unfolded state at low temperature is not unstructured. Molecular dynamics simulations at low temperature reveal a stretched-exponential behavior which originates from parallel folding routes that start from a kinetically partitioned unfolded ensemble. Different metastable structures (i.e., traps) in the unfolded ensemble have a different ratio of loop and helical content. Control simulations of the peptide at high temperature, as well as without the cross-linker at low temperature, show faster and simpler (i.e., single-exponential) folding kinetics. The experimental and simulation results together provide strong evidence that the rate-limiting step in formation of a structurally constrained α-helix is the escape from heterogeneous traps rather than the nucleation rate. This conclusion has important implications for an α-helical segment within a protein, rather than an isolated α-helix, because the cross-linker is a structural constraint similar to those present during the folding of a globular protein.

Ultrafast two dimensional-infrared spectroscopy of a molecular monolayer.

The study of vibrational coupling and energy flow in bulk (bio)molecular systems using two-dimensional infrared spectroscopy, has dramatically broadened our ability to elucidate structures and their dynamic evolution on ultrafast timescales. For molecules at surfaces, however, these insights have been lacking. In our study, vibrational coupling in a molecular monolayer is revealed by ultrafast two-dimensional vibrational spectroscopy, with interface specificity and (sub)monolayer sensitivity. This technique provides information on vibrational coupling and energy transfer at surfaces and interfaces with subpicosecond time resolution rendering it a unique tool for the investigation of both structural and dynamical surface processes in a wide variety of disciplines.

Interface-Specific Ultrafast Two-Dimensional Vibrational Spectroscopy

Surfaces and interfaces are omnipresent in nature. They are not just the place where two bulk media meet. Surfaces and interfaces play key roles in a diversity of fields ranging from heterogeneous catalysis and membrane biology to nanotechnology. They are the site of important dynamical processes, such as transport phenomena, energy transfer, molecular interactions, as well as chemical reactions. Tools to study molecular structure and dynamics that can be applied to the delicate molecular layers at surfaces and interfaces are thus highly desirable. The advent of multidimensional optical spectroscopies, which are the focus of a special issue of Accounts of Chemical Research, and in particular of two-dimensional infrared (2D-IR) spectroscopy has been a breakthrough in the investigation of ultrafast molecular dynamics in bulk media. This Account reviews our recent work extending 2D-IR spectroscopy to make it surface-specific, allowing us to reveal the structure and dynamics of specifically interfacial molecules. 2D-IR spectroscopy provides direct information on the coupling of specific vibrational modes. Coupling between different modes can be resolved and quantified by exciting a particular mode at a specific frequency and probing the effect of the excitation on a different mode at a different frequency. The response is thus measured as a function of two frequencies: the excitation and the probe frequency, which provides a two-dimensional vibrational spectrum. When two vibrational modes are coupled, this will give rise to the intensity in the off-diagonal part of the 2D-IR spectrum. The intensity of the cross-peak is determined by the strength of the coupling between the two modes, which, in turn, is determined by molecular conformation. One can therefore relate the 2D-IR spectrum to the molecular structure. By delaying pump and probe pulses relative to one another, one can obtain additional information about conformational fluctuations. The surface-specific 2D-IR approach presented here combines the virtues of 2D-IR with the surface specificity and sub-monolayer sensitivity of vibrational sum frequency generation (SFG). We demonstrate its application on a self-assembled monolayer of a primary alcohol on water. It allows for the elucidation of different contributions to the coupling between the different interfacial methyl and methylene stretching modes. Although the surface 2D-IR technique presented here is conceptually closely related to its bulk counterpart, it is shown to have distinct characteristics, owing to the preferential alignment of molecules at the interface and the strict selection rules of the SFG probing scheme. We present an analytic theoretical framework that incorporates these effects and present simulations on instructive examples as well as on the alcohol monolayer. Overall, these results illustrate the potential of extending 2D-IR spectroscopy to the investigation of surface molecular dynamics.

Protein ligand migration mapped by nonequilibrium 2D-IR exchange spectroscopy

2D-IR exchange spectroscopy has been introduced recently to map chemical exchange networks in equilibrium with subpicosecond time resolution. Here, we demonstrate the generalization of 2D-IR exchange spectroscopy to nonequilibrium systems and its application to map light-triggered migration of ligands between different sites in a protein. Within picoseconds after a photodissociating laser pulse, carbon monoxide ligands relocate from their binding site A at the heme prosthetic group of myoglobin to a primary docking site B in the distal heme pocket. Multiple CO stretching bands are observed for the CO ligand in sites A and B, indicating that several distinct conformational substates of the myoglobin:ligand complex coexist in solution. Exchange cross-peaks between the bands associated with substates of heme-bound CO and photodissociated CO in the primary docking site reveal the substate connectivity at physiological temperature.

Folding and unfolding of a photoswitchable peptide from picoseconds to microseconds

Using time-resolved IR spectroscopy, we monitored the kinetics of folding and unfolding processes of a photoswitchable 16-residue alanine-based α-helical peptide on a timescale from few picoseconds to almost 40 μs and over a large temperature range (279–318 K). The folding and unfolding processes were triggered by an ultrafast laser pulse that isomerized the cross linker within a few picoseconds. The main folding and unfolding times (700 ns and 150 ns, respectively, at room temperature) are in line with previous T-jump experiments obtained from similar peptides. However, both processes show complex, strongly temperature-dependent spectral kinetics that deviate clearly from a single-exponential behavior. Whereas in the unfolding experiment the ensemble starts from a well defined folded state, the starting ensemble in the folding experiment is more heterogeneous, which leads to distinctly different kinetics of the experiments, because they are sensitive to different regions of the energy surface. A qualitative agreement with the experimental data-set can be obtained by a model where the unfolded states act as a hub connected to several separated “misfolded” states with a distribution of rates. We conclude that a rather large spread of rates (k1 : kn ≈ 9) is needed to explain the experimentally observed stretched exponential response with stretching factor β = 0.8 at 279 K.

Versatile small volume closed-cycle flow cell system for transient spectroscopy at high repetition rates

A closed-cycle flow cell system was designed and built for application in ultrafast laser spectroscopy. The system features small cycle volume of down to less than 200 μl, adjustable beam path length, and compatibility with a large variety of solvents. The construction principle is simple, reliable, and versatile. The thickness and material of the windows can be easily exchanged to meet different demands of femtosecond spectroscopy. The flow rate is sufficient to allow complete sample exchange for measurements with a kHz repetition rate.

Peptide structure determination by two-dimensional infrared spectroscopy in the presence of homogeneous and inhomogeneous broadening

The potential information content of two-dimensional infrared (2D-IR) spectroscopy of the amide-I band as a structure analysis method of small peptides is explored in a computational study, applying it to a cyclic penta peptide as an example. In the presence of realistic homogeneous and inhomogeneous broadening, the structure resolution power in the case of a nonisotope labeled molecule would be vanishingly small. However, 2D-IR spectroscopy can reveal the structure of the peptide uniquely if using a sufficiently large set of isotope labeled compounds. Design strategies for isotope labeling are developed. In the case of the cyclic penta peptide studied here, at least three single 13C labeled compounds would be needed to determine the structure. While double 13C labeling does not offer any advantage compared to single 13C labeling, mixed 13C 16O–12C 18O or 13C 16O–13C 18O double labeling does. It is furthermore explored to what extent a structure can still be determined even under nonideal conditions, i.e....

Transient two-dimensional infrared spectroscopy: Exploring the polarization dependence

We present a general expression for the polarization dependence of transient two-dimensional IR spectroscopy (T2D-IR), a technique designed to measure 2D-IR spectra of transient species. T2D-IR is a UV pump narrowband-IR-pump broadband-IR-probe experiment of fifth order in the laser field which involves up to three different transition dipole moments. The UV pulse adds an additional degree of freedom in polarization as compared to 2D-IR spectroscopy and increases the versatility of signal manipulation and the potential structural information content of the signals. The polarization conditions leading to a maximum of structural information are discussed. Important special cases of polarization conditions are formulated. The application of polarization selectivity is demonstrated for different types of T2D-IR experiments on photo triggered metal-to-ligand charge transfer in the model system [Re(CO)(3)(dmbpy)Cl].