Application and Development of 2D IR Spectroscopy for the Study of Complex Biological Systems

Abstract

One of the many applications of 2D IR spectroscopy is in the study of structure/function relationships in proteins. While this technique can be used for structural characterization, the power of this tool is with its ability to provide timescales for the structural dynamics of a system in solution with picosecond time resolution. It is in this manner that 2D IR was used to understand the pH-dependent release mechanism of nitric oxide in the heme protein Nitrophorin-4. By probing the vibrational stretch of the nitrosyl ligand as a function of time, shifts of the vibrational stretch report time-dependent changes of the local electric field around the ligand and its interaction with the ferric heme center. The timescales extracted indicate that the ligand is sensitive to pH-dependent changes to the local distal pocket environment and to two distinct conformations of the protein. While this protein has numerous vibrational modes, the limited bandwidth (~250 cm<super>-1</super>) of the mid-IR source confined the spectral window to the nitrosyl stretch. To overcome this limit for future studies, a tunable and spectrally broad mid-IR pulse source was developed. This new source generates sub-20 fs, octave-spanning (> 2000 cm<super>-1</super>) broadband mid-IR pulses and is tunable from 2-8 μm. These mid-IR pulses are generated by four-wave mixing during the filamentation of intense 800 nm and 400 nm pulses in various gas media. Spectral tunability is achieved by the choice of gas, pressure and input 800 nm pulse energy. Pulses from this source would allow one to coherently probe numerous vibrations, potentially revealing correlations between local and global structural fluctuations.