RESUMO
Vibrational spectroscopy of protein structure often utilizes 13C18O-labeling of backbone carbonyls to further increase structural resolution. However, sidechains such as arginine, aspartate, and glutamate absorb within the same spectral region, complicating the analysis of isotope-labeled peaks. In this study, we report that the waiting time between pump and probe pulses in two-dimensional infrared spectroscopy can be used to suppress sidechain modes in favor of backbone amide I' modes based on differences in vibrational lifetimes. Furthermore, differences in the lifetimes of 13C18O-amide I' modes can aid in the assignment of secondary structure for labeled residues. Using model disordered and ß-sheet peptides, it was determined that while ß-sheets exhibit a longer lifetime than disordered structures, amide I' modes in both secondary structures exhibit longer lifetimes than sidechain modes. Overall, this work demonstrates that collecting 2D IR data at delayed waiting times, based on differences in vibrational lifetime between modes, can be used to effectively suppress interfering sidechain modes and further identify secondary structures.