Abstract2D IR spectroscopy was used to probe the hydrophobic core structure of the 35-residue Villin headpiece subdomain, HP35, by monitoring the C≡N vibrational stretching band of a cyano substituted phenylalanine (Phe). The presence of two humps in the vibrational frequency distribution in the folded equilibrium state is revealed. They represent two states that exchange more slowly than ca. 10 ps. The two CN stretch mode peak frequencies (and their equilibrium populations) are 2228.7 (44%) and 2234.5 cm −1 (56%). The two CN modes have different frequency-frequency correlation times of 7.4 ps and 1.6 ps respectively. These results suggest that the population with the higher frequency CN group is partly exposed whereas the other CN mode experiences a hydrophobic like environment. KeywordsNitrile probe; Two dimensional infrared spectroscopy; Villin headpiece; protein structural heterogeneity; cyanophenylalanineThe increasing availability of molecular dynamics simulations of protein structural fluctuations and of folding pathways 1-4 raises interesting questions about how the implied, detailed, microscopic predictions, which often involve ultrafast structural fluctuations, can be tested by real time experiments that monitor events that occur faster than the rate determining steps of conventional kinetics experiments. 5 Different peptide conformations necessarily have different dispositions of backbone and side chains so that they have different vibrational spectra. Two dimensional infrared spectroscopy (2D IR) 6-9 is a method of choice for examining protein structural constraints and their fluctuations over a broad time range. The peptide backbone amide groups have coupling parameters that are in principle deducible from these multidimensional infrared methods. The parameters of 2D IR in turn relate to the "instantaneous" backbone structure. The inverse bandwidth of commonly occurring vibrational transitions sets a time scale of a few picoseconds, such that conformational exchanges would have to occur faster than this to be motionally averaged. 10 Thus all the major structures anticipated in peptide conformational dynamics are expected to be spectrally distinct in 2D IR and with better resolution than FTIR.The dynamics within the vibrational frequency distribution of a mode, often referred to as spectral diffusion, signifies the time dependent structure of the immediate environment of the mode. This information is contained in the frequency-frequency correlation functions obtained from 2D IR. 9 Measurements of these correlations over a wide range of times reveal the dynamics local to an amide as well as those associated with the coupling between modes on different sections of the secondary structure. 9,11 These concepts apply equally well to the vibrational modes of probes that are engineered into the backbone or side chains of the peptides. The probes could be non-perturbative isotopologues or chemically modified residues as in the present paper.Nitrile groups have been very useful as vibrational probes of biolog...

The nearly degenerate asymmetric stretch vibrations near 1600 cm −1 of the guanidinium cation in D-glycerol/D 2 O mixtures having different viscosity were studied by 2D IR photon echo spectroscopy. The polarization dependent photon echo signal shows two separate frequency distributions in the 2D spectrum in D 2 O, even though only one band is evident from inspection of the linear FTIR spectrum. The split components are more clearly seen at higher viscosity. The interactions with solvent induce energy transfer between the degenerate component modes on the time scale of 0.5 ps. The energy transfer between modes is directly observed in 2D IR and distinguished by the waiting time dependence of the cross peaks from the transfers between threefold symmetric configurations of the distorted ion and solvent. The 2D IR analysis carried out for various polarization conditions required specification of frequency-frequency auto-and cross-correlation functions for the degenerate components.

Nearly degenerate asymmetric stretches with perpendicular transition dipole moments of the deuterated guanidinium cation (DGdm + ) in D 2 O and D-glycerol/D 2 O mixtures at 1600 cm −1 were investigated by linear FTIR spectroscopy and polarization dependent femtosecond pump-probe spectroscopy. The vibrational coupling of the asymmetric stretches of guanidinium occurs within 0.5 ps and leads to fast decay of the anisotropy to a level of 0.1. A systematic study of the influence of the coherence transfer on pump-probe signals is given. Following this decay the anisotropy decays with a time constant of 4.1 ps in D 2 O by rotational diffusion about an axis perpendicular to the DGdm + mean plane. The presence of aggregation was demonstrated for concentrations higher than 0.2 M.

The asymmetric stretching vibration of the amphiphilic trifluoroacetate ion and its 13 C v 16 O isotopologue in D 2 O were investigated with infrared spectroscopy ͑FTIR͒, ultrafast infrared pump probe, and two dimensional vibrational photon echo techniques and simulations. Trifluoroacetate ions have a nonexponential depopulation of the first vibrational excited state, which is well described by a kinetic mechanism involving a temperature dependent solvent assisted relaxation to the symmetric stretch mode. The vibrational spectrum of the asymmetric stretch of the 13 C v 16 O isotopologue presents an unusual spectral shape. The frequency-frequency autocorrelation function shows a static term not present in the 13 C v 16 O form, which is caused by an accidental degeneracy with a combinational mode. A newly developed frequency map for carboxylate is used to characterize the processes and dynamics observed in the frequency fluctuations of the carboxylate asymmetric stretch mode in aqueous solution. An assignment of the molecular processes that govern the frequency fluctuations is suggested from an analysis of the solvation shell configurations obtained from molecular dynamics simulations.

Dual frequency two-dimensional infrared spectroscopy (2D-IR) has been used to investigate the dynamics of the azide-water solvation shell. The memory of the azide transition frequencies is detected in the echo emitted by the OH stretching mode of the ion-bound water molecules. There is a significant positive correlation of the two frequency distributions that decays on a 140 fs time scale. The result confirms that the O-H bond of water molecules in the solvent shell have frequency fluctuations that are considerably slowed from those that are known in bulk water. The positive correlation is attributed to cooperative interactions of coordinated water molecules with an azide ion.