Proteins exhibit structural fluctuations over decades of time scales. From the picosecond side chain motions to aggregates that form over the course of minutes, characterizing protein structure over these vast lengths of time is important to understanding their function. In the past 15 years, two-dimensional infrared spectroscopy (2D IR) has been established as a versatile tool that can uniquely probe proteins structures on many time scales. In this review, we present some of the basic

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... s behind 2D IR and show how they have, and can, impact the field of protein biophysics. We highlight experiments in which 2D IR spectroscopy has provided structural and dynamical data that would be difficult to obtain with more standard structural biology techniques. We also highlight technological developments in 2D IR that continue to expand the scope of scientific problems that can be accessed in the biomedical sciences. Graphical Abstract INTRODUCTION The structural evolution of proteins, designed to accomplish precise functions, is at the heart many processes in biology. 1-3 Knowledge of the kinetics of the interconversion between In addition to the static distribution of local mode energies and structures (which create a distribution of couplings), these quantities can also be dynamic. That is, as we change the waiting time of the 2D IR spectrum, individual site energies and couplings could be modulated. This leads to a change in line shape in a 2D IR spectrum. This dynamic disorder is best described by a line shape function as opposed to generating many different Hamiltonians and adding some fluctuations about a mean value. We describe this in more detail in section 3.7. Polarization Controlled 2D IR To Determine Structure Since 2D IR is a coherent spectroscopy, all of the interacting fields have polarizations that can be independently controlled. Control of the polarization of the pump and probe fields Ghosh et al.