A model for the calculation of amide I FTIR and 2DIR spectra taking into account fluctuations in hydrogen bonding and structure from molecular dynamics (MD) simulations is tested on three systems. It is found that although the homogeneous lineshape approximation yields satisfactory FTIR spectra, 2DIR spectra are sensitive to the inhomogeneity naturally present in any solvated protein and the common approximations of a static structure and averaged-effect solvent are invalid. By building on the

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... ocal amide Hamiltonian and incorporating site energy variation with electrostatic-based models and disorder from MD trajectories, good agreement is obtained between calculated and measured 2DIR spectra. The largest contribution to the observed inhomogeneity is found to be the fluctuating site energies, which in turn are most sensitive to the water solvent. With the ability to accurately predict 2DIR spectra from atomistic simulations, new opportunities to test force fields and mechanistic predictions from MD are revealed.