Isotope-Edited Amide II Mode: A New Label for Site-Specific Vibrational Spectroscopy

Vibrational spectroscopy is a powerful tool used to analyze biological and chemical samples. However, in proteins, the most predominant peaks that arise from the backbone amide groups overlap one another, hampering site-specific analyses. Isotope editing has provided a robust, noninvasive approach to overcome this hurdle. In particular, the 1-C-13 = O-16 and 1-C-13 = O-18 labels that shift the amide I vibrational mode have enabled 1D- and 2D-IR spectroscopy to characterize proteins with excellent site-specific resolution. Herein, we expand the vibrational spectroscopy toolkit appreciably by introducing the 1-C-13-N-15 probe at specific locations along the protein backbone. A new, isotopically edited amide II peak is observed clearly in the spectra despite the presence of unlabeled modes arising from the rest of the protein. The experimentally determined shift of -30 cm(-1) is reproduced by DFT calculations providing further credence to the mode assignment. Since the amide II mode arises from different elements than the amide I mode, it affords molecular insights that are both distinct and complementary. Moreover, multiple labeling schemes may be used simultaneously, enhancing vibrational spectroscopy's ability to provide detailed molecular insights.

Automated summary

Vibrational spectroscopy is a powerful tool for analyzing biological and chemical samples. Isotope editing, particularly using 1-C-13 = O-16 and 1-C-13 = O-18 labels, has enabled excellent site-specific resolution in characterizing proteins. The introduction of the 1-C-13-N-15 probe along the protein backbone has expanded the vibrational spectroscopy toolkit, providing new molecular insights. The experimentally determined shifts and DFT calculations support the mode assignment, showing the potential for simultaneous use of multiple labeling schemes to enhance detailed molecular insights.