Water Penetration into Protein Secondary Structure Revealed by Hydrogen−Deuterium Exchange Two-Dimensional Infrared Spectroscopy

Two-dimensional infrared spectroscopy in conjunction with hydrogen−deuterium exchange experiments provides detailed information about solvent penetration into protein structure. Correlating the secondary-structure sensitivity of the amide I vibration and the solvent-exposure sensitivity of amide II provides a direct probe of solvent-inaccessible residues of proteins embedded in the hydrophobic core or those involved in strong hydrogen bonds in secondary structures. Distinct spectral signatures of the cross-peak region arising from the coupling of the amide I and II modes imply a significant degree of structural stability of hydrogen-bonded contacts in α-helices and β-sheets in a series of proteins. Ubiquitin, an α/β-protein, exhibits strong α-helical signatures and lacks those of the β-sheet in the cross-peak region, demonstrating that ubiquitin's β-sheet exchanges protons with the surrounding solvent and is conformationally unstable.