The N-H stretching vibration is an important probe for investigating structural and functional properties of proteins but is often difficult to analyze as it overlaps with the O-H stretching vibration of water molecules. In this work we investigate the N-H signals of hydrophobins using conventional (VSFG) and heterodyne-detected vibrational sum-frequency generation spectroscopy (HD-VSDG). Hydrophobins represent a group of surface active proteins that form highly-ordered protein films at the water-air interface and that give rise to prominent vibrational modes. We find that in conventional VSFG spectra N-H specific signals show significant changes in shape and intensity upon altering the pH values. These changes can easily be misinterpreted for conformational changes of the protein. Using HD-VSFG experiments, we demonstrate, that for hydrophobin films the change of the N-H response with pH can be well explained from the interference of the N-H response with the broad interfacial water O-H stretch band.
- air-water interface
- interfacial behavior
- vibrational sum-frequency generation spectroscopy
Meister, K., Paananen, A., & Bakker, H. (2017). Identification of the response of protein N-H vibrations in vibrational sum-frequency generation spectroscopy of aqueous protein films. Physical Chemistry Chemical Physics, 19(17), 10804-10807. https://doi.org/10.1039/C6CP08325K