Vibrational dynamics in peptides and proteins using two dimensional infrared spectroscopy

The research presented in this thesis advances the applicability of two dimensional infrared spectroscopic methods to probe the dynamics of local environments of peptides and proteins at equilibrium. 2D IR experiments described herein investigated the hydrogen bond dynamics of two aromatic nitriles in methanol and established the experimental parameters for utilizing nitriles as probes of solvent environments in complex biomolecules. Cyano-phenylalanine mutants of the M2 influenza channel were employed to assess pH induced hydration changes near the tryptophan gate that controls flow of water through the channel. A structurally less perturbative approach of utilizing isotopically substituted backbone amide vibrations to probe water structures was also evaluated. The lessons learned through this exercise were applied to exploring the ebb and flow of water in the M2 channel through the 2D IR spectral dynamics of the amide mode of the Gly34 residue. The 2D IR spectroscopy of Gly34 in the M2 channel revealed that the conformational equilibrium in M2 entails a change in the mobility of the channel water similar to what might be expected for phase transition from frozen to liquid water. The aforementioned approach was extended to address drug binding modes in the channel. 2D IR experiments with drug-free and drug-bound channels indicated that the water cluster near Gly34 is unperturbed when less bulky drugs such as rimantadine bind in the channel, whereas, binding of bulkier drug such as spiran amine affords increased mobility in the water cluster sensed by Gly34, which is reflected in the spectral densities of the Gly34 amide, which is in qualitative consistency with the model proposed from MD simulations. The investigation of isotopically labeled amide vibrations involved examination of sidechain transitions that absorb in the same spectral range. The sidechain modes of the guanidyl moiety of arginine was examined, and 2D IR spectral evolution revealed ultrafast energy transfer occurring between the nearly degenerate modes of the guanidyl sidechain, thus forwarding the spectral dynamics as an added dimension for characterizing vibrations in proteins.