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We have investigated the site-specific folding kinetics of a photo-switchable cross-linked [alpha]-helical peptide by using single [sup.13]C = [sup.18]O 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[degrees]C), whereas at high temperatures (45[degrees]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 [alpha]-helix is the escape from heterogeneous traps rather than the nucleation rate. This conclusion has important implications for an [alpha]-helical segment within a protein, rather than an isolated [alpha]-helix, because the cross-linker is a structural constraint similar to those present during the folding of a globular protein. cooperativity | infrared spectroscopy | molecular dynamics simulation | peptide folding

Two photocleavable disulfide-bridged tetrapeptides [cyclo(Boc-Cys-Pro-Aib-Cys-OMe) and cyclo(Boc-Cys-Pro-Phe-Cys-OMe)] which provide insight into cyclic peptide dynamics are reported.

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. ligand binding | myoglobin | chemical exchange | infrared spectroscopy

We investigate energy transport through an [alpha]-aminoisobutyric acid-based [3.sub.10]-helix dissolved in chloroform in a combined experimental-theoretical approach. Vibrational energy is locally deposited at the N terminus of the helix by ultrafast internal conversion of a covalently attached, electronically excited, azobenzene moiety. Heat flow through the helix is detected with subpicosecond time resolution by employing vibrational probes as local thermometers at various distances from the heat source. The experiment is supplemented by detailed nonequilibrium molecular dynamics (MD) simulations of the process, revealing good qualitative agreement with experiment: Both theory and experiment exhibit an almost instantaneous temperature jump of the reporter units next to the heater which is attributed to the direct impact of the isomerizing azobenzene moiety. After this impact event, helix and azobenzene moiety appear to be thermally decoupled. The energy deposited in the helix thermalizes on a subpicosecond timescale and propagates along the helix in a diffusive-like process, accompanied by a significant loss into the solvent. However, in terms of quantitative numbers, theory and experiment differ. In particular, the MD simulation seems to overestimate the heat diffusion constant (2 [[Angstrom].sup.2] [ps.sup.-1] from the experiment) by a factor of five. energy dissipation | time-resolved IR spectroscopy | peptide helix | nonequilibrium molecular dynamics simulation

Using time-resolved IR spectroscopy, we monitored the kinetics of folding and unfolding processes of a photoswitchable 16-residue alanine-based [alpha]-helical peptide on a timescale from few picoseconds to almost 40 [micro]s and over a large temperature range (279-318K). 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 ([k.sub.1]: [k.sub.n] [approximately equal to]9) is needed to explain the experimentally observed stretched exponential response with stretching factor [bata] = 0.8 at 279 K.

Author(s): Christoph Kolano (corresponding author) [1]; Jan Helbing [1]; Mariusz Kozinski [1]; Wolfram Sander [2]; Peter Hamm (corresponding author) [1] X-ray crystallography and nuclear magnetic resonance measurements provide us with [...]

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 [micro]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 l' 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 l' band, isotope-labeled amino acid residues, and side chains. We find differing relaxation dynamics at different spectral positions. [alpha]-helix folding | femtosecond IR spectroscopy | protein folding

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.

Observation of coherent vibrational dynamics in a hydrogen bond obtained by femtosecond nonlinear measurements in mid-infrared spectral range is reported. The result provide experimental evidence in the time domain for the anharmonic coupling of slow and fast vibrational motions in hydrogen bond.

Stimulated photon echo experiments were used to study the amide I band region of globular peptides, apamin and a cyclic pentapeptide.

A form of two-dimensional (2D) vibrational spectroscopy, which uses two ultrafast IR laser pulses, is used to examine the structure of a cyclic penta-peptide in solution. Spectrally resolved cross peaks occur in the off-diagonal region of the 2D IR spectrum of the amide I region, analogous to those in 2D NMR spectroscopy. These cross peaks measure the coupling between the different amide groups in the structure. Their intensities and polarizations relate directly to the three-dimensional structure of the peptide. With the help of a model coupling Hamiltonian, supplemented by density functional calculations, the spectra of this penta-peptide can be regenerated from the known solution phase structure. This 2D-IR measurement, with an intrinsic time resolution of less than 1 ps, could be used in all time regimes of interest in biology.

The correlation functions of the fluctuations of vibrational frequencies of azide ions and carbon monoxide in proteins are determined directly from stimulated photon echoes generated with femtosecond infrared pulses. The asymmetric stretching vibration of azide bound to carbonic anhydrase II exhibits a pronounced evolution of its vibrational frequency distribution on the time scale of a few picoseconds, which is attributed to modifications of the ligand structure through interactions with the nearby Thr-199. When azide is bound in hemoglobin, a more complex evolution of the protein structure is required to interchange the different ligand configurations, as evidenced by the much slower relaxation of the frequency distribution in this case. The time evolution of the distribution of frequencies of carbon monoxide bound in hemoglobin occurs on the [approximately equal to]10-ps time scale and is very nonexponential. The correlation functions of the frequency fluctuations determine the evolution of the protein structure local to the probe and the extent to which the probe can navigate those parts of the energy landscape where the structural configurations are able to modify the local potential energy function of the probe.

The time-resolved femtosecond spectroscopy in conjunction with nonequilibrium molecular dynamics (MD) simulations was used to study the energy transport in a short helical peptide in chloroform solution. A simple harmonic model of heat transport adopted on the bases of results showed that the heat diffusivity decreased significantly at temperatures initially reached by high-energy excitation.

The potential of transient two-dimensional (2D) infrared spectroscopy is demonstrated to resolve the vibrational bands hidden in conventional pump-probe spectra of the thioxopeptide Boc-Ala-Gly([double bond]S)-Ala-Aib-OMe. Different types of transient cross-peak signals are seen and their information is discussed by comparing with model spectra.

The two-dimensional infrared (2D-IR) spectroscopy technique is employed to study the ultrafast structural dynamics of the photoswitchable peptides. The mechanism involved in the unfolding of the peptide secondary structure motifs is also discussed.

Time-resolved infrared spectroscopy was used to study the photoisomerization of N-Methylthioacetamide (NMTAA) in D2O in both the cis-> trans and the trans->cis direction upon selective excitation of the n-pi* (S1) and pi-pi* (S2) electronic transitions. Results support a mechanism by which isomerization takes place via one common intermediate state independent of electronic excitation energy and initial conformation.

Thio amino acids can be infused into the essence of peptides without excessively disturbing their structure. The photoisomerization of the trans form of N-methylthioacetamide as a model conformational photoswtich is examined.

Ultrafast two-dimensional infrared spectroscopy (2D-IR) is a promising tool to investigate molecular structure. The investigation of this conformational transition of a photoswitchable cyclic peptide by transient 2D-IR spectroscopy (T2D-IR) is done.

Transient 2D infrared (two-dimensional infrared), (T2D-IR), snapshots of the peptide ensemble taken during the transition from the cis to the trans configuration are presented. The results suggest that the homogenous to the total width of the amide-I band changes during the course of the conformational transition.

The observation of subpicosecond fluctuations in the conformation of a small peptide in water is demonstrated. We use an experimental method that is specifically sensitive to conformational dynamics taking place on an ultrafast time scale. Complementary molecular-dynamics simulations confirm that the conformational fluctuations exhibit a subpicosecond component, the time scale and amplitude of which agree well with those derived from the experiment.