Two dimensional infrared (2D IR) spectroscopy has been successfully adapted and applied to the coupled amide-I modes to obtain a detailed understanding of the structures, interactions and conformational dynamics of peptides and proteins in solution. The structure was estimated from the polarization dependent 2D IR experiments and the anharmonic coupling between the amide I modes. The interaction was directly monitored from the cross peaks arising due to coupling between the amide-I modes and the ionizable side chains. The equilibrium kinetics of the conformational transitions was quantitatively estimated from the growth of the cross peaks between distinct vibrational transitions of the conformers as a function of the population period.
The linear infrared and 2D IR spectra in the amide-I region of N-acetyl tryptophan methyl amide (NATMA) in solvents of varying polarity are reported. The major conformer in CH 2 Cl 2 corresponds to a C 7 structure, in agreement with that found in the gas phase with intramolecular hydrogen bonding between the acetyl end C=0 and the amino end N-H.
The study of sub-nanosecond equilibrium conformational transitions of NATMA by 2D IR has been reported. Multiple transitions are observed for the acetyl end amide-I mode indicating existence of multiple conformers in D 2 O. A detailed study of the waiting time dependence of the intramode cross peaks shows evidence of interchanging conformational distributions. Molecular Dynamics (MD) simulations complements the experimental results by predicting two conformational distributions resulting significantly from the C α -C β bond rotation. The hydrogen bond dynamics of the acetyl end carbonyl and the surrounding water molecules has been estimated from the MD simulations
The interaction between the ionizable carboxylate side-chains and the amide-I modes of Villin headpiece has been investigated. The transition frequencies of the carboxylate groups spectrally overlap with the isotopically labeled amide-I transition frequency region. Theoretical calculations show these side chains to interact with only a few amide-I modes and thus may be useful as spectroscopic markers to probe the site specific local structural behavior of the protein.
