How to detect the functionally important structural motions of proteins, including proton transfer, electron transfer, changes in active site hydrogen bonding interactions, formation and cleavage of covalent bonds, and changes in protein folding from picoseconds to seconds? We are developing infrared structural biology as a major technique for dynamic structural studies of protein functions.We focus on a critical issue, how to translate infrared spectral signals into specific structural data.Here we report the development of vibrational structural markers (VSM)for probing hydrogen bonding interactions of buried COO-(Asp/Glu) in proteins.Quantum theory based first principle computational studies combined with FTIR studies and bioinformatics analysis were employed in this study.Infrared structural biology is expected to emerge as a powerful technique for exploring the functional mechanism of a broad range of proteins.