We have studied the structure of (Ala)5, a model unfolded peptide, using a combination of 2D IR spectroscopy and molecular dynamics (MD) simulation. Two different isotopomers, each bis-labeled with 13C═O and 13C═18O, were strategically designed to shift individual site frequencies and uncouple neighboring amide-I′ modes. 2D IR spectra taken under the double-crossed ⟨π/4, –π/4, Y, Z⟩ polarization show that the labeled four-oscillator systems can be approximated by three two-oscillator systems. By utilizing the different polarization dependence of diagonal and cross peaks, we extracted the coupling constants and angles between three pairs of amide-I′ transition dipoles through spectral fitting. These parameters were related to the peptide backbone dihedral angles through DFT calculated maps. The derived dihedral angles are all located in the polyproline-II (ppII) region of the Ramachandran plot. These results were compared to the conformations sampled by Hamiltonian replica-exchange MD simulations with three different CHARMM force fields. The C36 force field predicted that ppII is the dominant conformation, consistent with the experimental findings, whereas C22/CMAP predicted similar population for α+, β, and ppII, and the polarizable Drude-2013 predicted dominating β structure. Spectral simulation based on MD representative conformations and structure ensembles demonstrated the need to include multiple 2D spectral features, especially the cross-peak intensity ratio and shape, in structure determination. Using 2D reference spectra defined by the C36 structure ensemble, the best spectral simulation is achieved with nearly 100% ppII population, although the agreement with the experimental cross-peak intensity ratio is still insufficient. The dependence of population determination on the choice of reference structures/spectra and the current limitations on theoretical modeling relating peptide structures to spectral parameters are discussed. Compared with the previous results on alanine based oligopeptides, the dihedral angles of our fitted structure, and the most populated ppII structure from the C36 simulation are in good agreement with those suggesting a major ppII population. Our results provide further support for the importance of ppII conformation in the ensemble of unfolded peptides.

We have used a combination of 2D IR spectroscopy with 13C═18O labeled amide-I and 15N-labeled amide-II modes to reveal how vibrational coupling between labeled peptide units depends on secondary structure. Linear and 2D IR measurements and simulations of Cα,α-diethylglycine homotetrapeptide show that this compound adopts the fully extended (2.05-helical) conformation in CDCl3, consistent with previous work on the Ac-capped peptide. The amide-I/II cross peaks of isotopomers exhibit only a marginal isotope frequency shift between labeled modes that are separated by two peptide units, indicating a very weak coupling. This result is in sharp contrast with a large cross-peak shift observed in 310-helical peptides, in which the labeled amide-I and -II modes are connected through an inter-residue C═O···H–N hydrogen bond. The discovered 3D-structural dependence indicates that the 13C═18O/15N labeled amide-I/II cross peaks can distinguish the formation of a single 310-helical turn from the fully extended polypeptide chain and increase the versatility of 2D IR spectroscopy as a conformational analysis tool of biomolecules.

We demonstrate a phase sensitive, vibrationally resonant sum-frequency generation (PSVR-SFG) microscope that combines high resolution, fast image acquisition speed, chemical selectivity, and phase sensitivity. Using the PSVR-SFG microscope, we generate amplitude and phase images of the second-order susceptibility of collagen I fibers in rat tail tendon tissue on resonance with the methylene vibrations of the protein. We find that the phase of the second-order susceptibility shows dependence on the effective polarity of the fibril bundles, revealing fibrous collagen domains of opposite orientations within the tissue. The presence of collagen microdomains in tendon tissue may have implications for the interpretation of the mechanical properties of the tissue.

Molecular conformations around the C═O group of carbonyl compounds like ketones and aldehydes play an important role in determining their reaction properties in solutions, including reaction rate, mechanism, steric structure, and chirality of products. Investigating different rotational conformers and their rapid exchange at room temperature will provide information on the rotational barrier and insights into how different rotamers may contribute to fundamental reactions in chemistry. We applied two-dimensional infrared (2D IR) spectroscopy and polarization-dependent IR transient grating technique to the study of 4,4-dimethyl-2-pentanone in CCl4. Spectroscopic evidence showed that the internal rotation around the single carbon–carbon bond adjacent to the C═O group takes place on a picosecond time scale. DFT calculations suggested the presence of three different rotational conformations, one eclipsed and two staggered forms. Spectral simulation utilized the stochastic Liouville equation with a three-state jump model and incorporated the polarization factors that take into account the different direction of transition dipole moment in the three rotamers. The effects of the intramolecular vibrational energy redistribution process on the waiting time dependence of the 2D absorptive spectra were also included. Through comprehensive simulation of the observed spectral features, the exchange time constants between the three rotamers were determined: 5.4 ps from the eclipsed to staggered forms and 1.7 ps for the reverse direction.

We have carried out structural determination of capped Cα,α-diethylglycine (Deg) homopeptides with different chain lengths, Ac-(Deg)n-OtBu (n = 2−5), solvated in CDCl3, and investigated vibrational properties of the amide I and II modes by linear and 2D IR spectroscopy, ONIOM calculations, and molecular dynamics simulations. 2D IR experiments were performed in the amide I region using the rephasing pulse sequence under the double-crossed polarization and the nonrephasing sequence under a new polarization configuration to measure cross-peak patterns in the off-diagonal regions. The 2D IR spectra measured in the amide I and II regions reveal complex couplings between these modes. Model spectral calculations finely reproduced the measured spectral profiles by using vibrational parameters that were very close to the values predicted by the ONIOM method. The agreement led to a conclusion that peptide backbones are fully extended with the dihedral angles (ϕ,ψ) ≈ (±180°,±180°) and that a sequence of intramolecular C5 hydrogen bonds forms along the entire chain regardless of the chain length. This conclusion was endorsed by analysis of the molecular dynamics trajectories for n = 3 and 5 that showed an exclusive population of the C5 conformation. The conformationally well-restrained Deg homopeptides serve as an ideal linear exciton chain, which is scarcely obtainable by protein amino acids. We investigated excitonic properties of the linear chain through analytic modeling and compared the measurement and calculation results of the amide I and II modes. The integrated intensity of the amide II band is larger than that of the amide I for the C5 structure, untypical behavior in contrast with other secondary structures. This comprehensive study characterized the amide I and II spectral signatures of the fully extended conformation, which will facilitate the conformational analysis of artificial oligopeptides that contain such structural motifs.

We have carried out a comparative study of five ab initio electrostatic frequency maps and a semiempirical model for the amide-I and -II modes. Unrestrained molecular dynamics simulation of a 310-helical peptide, Z-Aib-l-Leu-(Aib)2-Gly-OtBu, in CDCl3 is performed using the AMBER ff99SB force field, and the linear and two-dimensional infrared (2D IR) spectra are simulated on the basis of a vibrational exciton Hamiltonian model. A new electrostatic potential-based amide-I and -II frequency map for N-methylacetamide is developed in this study. This map and other maps developed by different research groups are applied to calculate the local mode frequencies of the amide linkages in the hexapeptide. The simulated amide-I line shape from all models agrees well with the previous experimental results on the same system, except for an overall frequency shift. In contrast, the simulated amide-II bands are more sensitive to the frequency maps. Essential features obtained in the electrostatic models are captured by the semiempirical model that takes into account only the intramolecular hydrogen bonding effects and solvent shifts. Detailed comparisons between the models are also drawn through analysis of the local mode frequency shifts. Among all of the maps tested in this study, the new four-site potential map performs quite well in simulating the amide-II bands. It properly predicts the effects of hydrogen bonding on the amide-I and -II frequencies and reasonably simulates the isotope-dependent amide-I/II cross peaks upon 13C═18O/15N substitutions.

The interactions of neuropeptides and membranes play an important role in peptide hormone function. Our current understanding of peptide−membrane interactions remains limited due to the paucity of experimental techniques capable of probing such interactions. In this work, we study the nature of opioid peptide−membrane interactions using ultrafast two-dimensional infrared (2D IR) spectroscopy. The high temporal resolution of 2D IR is particularly suited for studying highly flexible opioid peptides. We investigate the location of the tyrosine (Tyr) side chain of leucine-enkephalin (Lenk) in lipid bilayer membranes by measuring spectral diffusion of the phenolic ring vibrational mode in three different systems: Lenk in lipid bilayer membranes (bicelles), Lenk in deuterated water, and p-cresol in deuterated water. Frequency−frequency correlation functions obtained from waiting-time-dependent 2D IR spectra reveal an ultrafast decaying component with an ∼1 ps time constant that is common for all three systems. On the basis of density functional theory calculations and molecular dynamics simulations, this spectral diffusion component is attributed to hydrogen-bond dynamics of the phenolic hydroxyl group interacting with bulk water. Unlike p-cresol in water, both Lenk systems exhibit static spectral inhomogeneity, which can be attributed to conformational distributions of Lenk that do not interconvert within 4 ps. Our results suggest that the Tyr side chain of Lenk in bicelles is located at the water-abundant region at the membrane−water interface and not embedded into the hydrophobic core.

We have combined two-dimensional infrared (2D IR) spectroscopy and isotope substitutions to reveal the vibrational couplings between a pair of amide-I and -II modes that are several residues away but directly connected through a hydrogen bond in a helical peptide. This strategy is demonstrated on a 310-helical hexapeptide, Z-Aib-l-Leu-(Aib)2-Gly-Aib-OtBu, and its 13C═18O-Leu monolabeled and 13C═18O-Leu/15N-Gly bis-labeled isotopomers in CDCl3. The isotope-dependent amide-I/II cross peaks clearly show that the second and fourth peptide linkages are vibrationally coupled as they are in proximity, forming a 310-helical turn. The experimental spectra are compared to simulations based on a vibrational exciton Hamiltonian model that fully takes into account the amide-I and -II modes. The amide-II local mode frequency is evaluated by a new model based on the effects of hydrogen-bond geometry and sites. Ab initio nearest-neighbor coupling maps of the amide-I/I, -I/II, -II/I and -II/II modes are generated by isotopically isolating the local modes of N-acetyl-glycine N′-methylamide (AcGlyNHMe). Longer range couplings are modeled by transition charge interactions. The effects of the capping groups are incorporated and isotope effects are analyzed based on ab initio calculations of six model compounds. The main features of the 2D IR spectra are reproduced by this modeling. The conformational sensitivity of the isotope-dependent amide-I/II cross peaks is discussed in comparison with the calculated spectra for a semiextended structure. Our experimental and theoretical study demonstrates that the combination of 2D IR and 13C═18O/15N labeling is a useful structural method for detecting helical turn formation with residue-level specificity.

We have investigated the sensitivity of two-dimensional infrared (2D IR) spectroscopy to peptide helicity with an experimental and theoretical study of Z-[l-(αMe)Val]8-OtBu in CDCl3. 2D IR experiments were carried out in the amide-I region under the parallel and the double-crossed polarization configurations. In the latter polarization configuration, the 2D spectra taken with the rephasing and nonrephasing pulse sequences exhibit a doublet feature and a single peak, respectively. These cross-peak patterns are highly sensitive to the underlying peptide structure. Spectral calculations were performed on the basis of a vibrational exciton model, with the local mode frequencies and couplings calculated from snapshots of molecular dynamics (MD) simulation trajectories using six different models for the Hamiltonian. Conformationally variant segments of the MD trajectory, while reproducing the main features of the experimental spectra, are characterized by extraneous features, suggesting that the structural ensembles sampled by the simulation are too broad. By imposing periodic restraints on the peptide dihedral angles with the crystal structure as a reference, much better agreement between the measured and the calculated spectra was achieved. The result indicates that the structure of Z-[l-(αMe)Val]8-OtBu in CDCl3 is a fully developed 310-helix with only a small fraction of α-helical or nonhelical conformations in the middle of the peptide. Of the four different combinations of pulse sequences and polarization configurations, the nonrephasing double-crossed polarization 2D IR spectrum exhibits the highest sensitivity in detecting conformational variation. Of the six local mode frequency models tested, the electrostatic maps of Mukamel and Cho perform the best. Our results show that the high sensitivity of 2D IR spectroscopy can provide a useful basis for developing methods to improve the sampling accuracy of force fields and for characterizing the relative merits of the different protocols for the Hamiltonian calculation.