The transition path is the tiny fraction of an equilibrium molecular trajectory when a transition occurs by crossing the free-energy barrier between two states. It is a single-molecule property that contains all the mechanistic information on how a process occurs. As a step toward observing transition paths in protein folding we determined the average transition-path time for a fast- and a slow-folding protein from a photon-by-photon analysis of fluorescence trajectories in single-molecule Förster-resonance-energy-transfer experiments. While the folding rate coefficients differ by a factor of 10,000, the transition-path times differ by less than a factor of 5, showing that a fast-and a slow-folding protein take almost the same time to fold when folding actually happens. A very simple model based on energy landscape theory can explain this result.

We review two-dimensional infrared (2D IR) spectroscopy of the amide I protein backbone vibration. Amide I modes are known for secondary structural sensitivity derived from their protein-wide delocalization. However, amide I FTIR spectra often display little variation for different proteins due to the broad and featureless line shape that arises from different structural motifs. 2D IR offers increased structural resolution by spreading the spectra over a second frequency dimension to reveal two-dimensional line shapes and cross-peaks. In addition, it carries picosecond time resolution, making it an excellent choice for understanding protein dynamics. In 2D IR spectra, cross peaks arise from anharmonic coupling between vibrations. For example, the spectra of ordered antiparallel beta sheets shows a cross peak between the strong nu perpendicular mode at approximately 1620 cm(-1) and the weaker nu parallel mode at approximately 1680 cm(-1). In proteins with beta-sheet content, disorder spreads the cross peaks into ridges, which gives rise to a "Z"-shaped contour profile. 2D IR spectra of alpha helices show a flattened "figure-8" line shape, and random coils give rise to unstructured, diagonally elongated bands. A distinguishing quality of 2D IR is the availability of accurate structure-based models to calculate spectra from atomistic structures and MD simulations. The amide I region is relatively isolated from other protein vibrations, which allows the spectra to be described by coupled anharmonic local amide I vibrations at each peptide unit. One of the most exciting applications of 2D IR is to study protein unfolding dynamics. While 2D IR has been used to study equilibrium structural changes, it has the time resolution to probe all changes resulting from photoinitiated dynamics. Transient 2D IR has been used to probe downhill protein unfolding and hydrogen bond dynamics in peptides. Because 2D IR spectra can be calculated from folding MD simulations, opportunities arise for making rigorous connections. By introduction of isotope labels, amide I 2D IR spectra can probe site-specific structure with picosecond time resolution. This has been used to reveal local information about picosecond fluctuations and disorder in beta hairpins and peptides. Multimode 2D IR spectroscopy has been used to correlate the structure sensitivity of amide I with amide II to report on solvent accessibility and structural stability in proteins.

We investigate the sensitivity of femtosecond Fourier transform two-dimensional infrared spectroscopy to protein secondary structure with a study of antiparallel beta-sheets. The results show that 2D IR spectroscopy is more sensitive to structural differences between proteins than traditional infrared spectroscopy, providing an observable that allows comparison to quantitative models of protein vibrational spectroscopy. 2D IR correlation spectra of the amide I region of poly-l-lysine, concanavalin A, ribonuclease A, and lysozyme show cross-peaks between the IR-active transitions that are characteristic of amide I couplings for polypeptides in antiparallel hydrogen-bonding registry. For poly-l-lysine, the 2D IR spectrum contains the eight-peak structure expected for two dominant vibrations of an extended, ordered antiparallel beta-sheet. In the proteins with antiparallel beta-sheets, interference effects between the diagonal and cross-peaks arising from the sheets, combined with diagonally elongated resonances from additional amide transitions, lead to a characteristic "Z"-shaped pattern for the amide I region in the 2D IR spectrum. We discuss in detail how the number of strands in the sheet, the local configurational disorder in the sheet, the delocalization of the vibrational excitation, and the angle between transition dipole moments affect the position, splitting, amplitude, and line shape of the cross-peaks and diagonal peaks.