Accurate determination of protein structure at interfaces
is critical
for understanding protein interactions, which is directly relevant
to a molecular-level understanding of interfacial proteins in biology
and medicine. Vibrational sum frequency generation (VSFG) spectroscopy
is often used for probing the protein amide I mode, which reports
protein structures at interfaces. Observed peak shifts are attributed
to conformational changes and often form the foundation of hypotheses
explaining protein working mechanisms. Here, we investigate structurally
diverse proteins using conventional and heterodyne-detected VSFG (HD-VSFG)
spectroscopy as a function of solution pH. We reveal that blue-shifts
of the amide I peak observed in conventional VSFG spectra upon lowering
the pH are governed by the drastic change of the nonresonant contribution.
Our results highlight that connecting changes in conventional VSFG
spectra to conformational changes of interfacial proteins can be arbitrary,
and that HD-VSFG measurements are required to draw unambiguous conclusions
about structural changes in biomolecules.