Vibrational dynamics in proteins using two-dimensional infrared spectroscopy

The time course of a vibrational probe is ultra-sensitive to the motions of nearby atoms, particularly those with net charges like water, which cause instantaneous fluctuations of the vibrational frequency. Two dimensional infrared spectroscopy (2D IR) leads to direct quantitative inferences on these motions. Over the past decade, 2D IR spectroscopy has developed into a promising method for probing site-specific structure and dynamics of peptides, proteins and other biological assemblies. The principles of 2D IR spectroscopy and approaches for extracting the vibrational frequency correlation function from 2D spectra will be discussed. The application of 2D IR methodologies, employed to investigate the pH induced ebb and flow of water in the M2 proton channel in influenza viruses through the spectral dynamics of the backbone amide modes, will be presented. The 2D IR spectroscopy of pore lining amides in the M2 channel reveal 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. This approach was extended to address drug binding modes in the channel. 2D IR experiments with drug-free and drug-bound channels expose water mobility in the channel under different drug binding conditions, which is reflected in the spectral dynamics of the Ala30 and Gly34 amides. The results suggest a favorable entropic factor for drug binding owing to disruption of water structures, thus revealing a functional model of drug binding in the channel that is in qualitative consistency with the model proposed from MD simulations. The unique capabilities of Fourier transform infrared spectroscopy can be utilized for imaging applications, and a new table-top technique for collecting wide-field Fourier transform infrared (FTIR) microscopic images by combining a femtosecond pulse shaper with a mid-IR focal plane array will be presented. Infrared absorption images were collected for a mixture of W(CO)6 or Mn2(CO)10 absorbed polystyrene beads, demonstrating that this technique can spatially resolve chemically distinct species. Extension of this method to hyperspectral 2D IR microscopy will also be discussed.