Two-dimensional Infrared Spectroscopy as a General Approach for the Study of Protein Dynamics

Book excerpt: Complete understanding of protein function requires knowledge of protein conformational dynamics, or the structural fluctuations of a protein. However, characterization of protein dynamics is challenged by protein complexity, as they are large, heterogeneous molecules with potentially important motions on very fast timescales. This complexity demands the use of a technique with high spatial and temporal resolution. Two-dimensional infrared (2D IR) spectroscopy has emerged as a powerful tool for the characterization and direct measurement of molecular heterogeneity and dynamics due to its excellent spatial and temporal resolution. However, application to proteins is hindered by their severely congested spectra due to the large number of similar bonds. To overcome this issue, proteins can be site-specifically labeled with spectrally resolved IR probes that are active in the transparent frequency region (~1800 - 2500 cm-1) and are sensitive to their environment. The studies presented here take advantage of the combination of site-specific labeling and IR spectroscopy to study the environments and dynamics at specific locations in three distinct protein systems. Herein, I describe our investigations of dynamic complexes of proteins that have challenged experimental characterization with conventional methods: plastocyanin (Pc) and its binding partner cytochrome f (cyt f); cytochrome P450cam (P450cam) and substrates or its redox partner, putidaredoxin; and the SH3Sho1 domain and the proline-rich (PR) recognition motif of its binding partner Pbs2. In addition, we describe my attempts at improving the experimental technique of site-specific IR spectroscopy as a general biophysical approach for protein characterization. Overall, I present evidence for the importance of fast dynamics in protein function and illustrate the rich information provided by 2D IR spectroscopy to complement existing biophysical methods.