Isotope Labeling of Small Peptides

The amide I band, near 1650 cm-1, arises primarily from carbonyl stretching modes in the peptide backbone and is often used to study protein secondary structures. The amide I band is sensitive to secondary structure, because the amide I vibrations from different amino acids are coupled to one another, creating delocalized modes whose frequencies depend on the geometry of the backbone.

To obtain information at a particular location in a protein rather than about the overall structure, we use isotope labeling. We synthesize peptides with one, or in some cases, two, 13C=18O isotope labels in order to determine the vibrational dynamics, relaxation, and coupling of specific residues through the polypeptide or protein. The 13C=18O label is preferable to the 13C label, because it causes a frequency shift of roughly 60 cm-1, allowing it to be resolved separately from the unlabeled amino acids.

We have applied this approach to study the CD3ζ transmembrane peptide, the M2 channel, the ovispirin anti-microbial peptide, and aggregating peptides. To synthesize these peptides, we purchase the 13C labeled amino acid and perform the 18O exchange by either acid-catalyzed oxygen exchange in H218O/dioxane or carbodiimide chemistry. We then synthesize the peptides using standard solid-phase peptide synthesis techniques.

Selected Publications
Marek P.; Woys A.M.; Sutton K.; Zanni M.T.; Raleigh D.P. Efficient Microwave-Assisted Synthesis of Human Islet Amyloid Polypeptide Designed to Facilitate the Specific Incorporation of Labeled Amino Acids. Org. Lett. 2010, 12(21), 4848.
Middleton, C. T.; Woys, A. M.; Mukherjee, S. S.; Zanni, M. T. Residue-Specific Structural Kinetics of Proteins through the Union of Isotope Labeling, Mid-IR Pulse Shaping, and Coherent 2D IR Spectroscopy. Methods 2010, 52, 12–22.