Chemical shift differences between free and Fab-bound peptide correlate with a two-stage selection of peptide sequences from a random phage display library to delineate critical and non-critical residues for antibody recognition.
Epitope libraries provide a method to identify peptide ligands for antibodies, receptors or other binding proteins. As such, they provide a powerful tool to rapidly identify lead ligands in the drug discovery process. In an attempt to correlate structural information with the results from peptide screening, we have used NMR spectroscopy of peptide/antibody complexes to demonstrate that core residues identified through a two-stage selection process undergo a larger structural change upon binding antibody than do positions in the peptide amenable to a variety of side chains. The model system used was the M2 monoclonal antibody/Flag octapeptide epitope system. We have analyzed two peptides: Ac-Asp-Tyr-Lys-Leu-Gly-Asp-Asp-Leu-NH2 (peptide 1), which contains several non-core positions randomized, and Ac-Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Leu-NH2 (peptide 2), which closely corresponds to the original Flag sequence. Enrichment of the peptides with 15N facilitated the investigation by permitting spectral editing of the peptide resonances in the presence of antibody. For peptide 1 the absolute shifts for the free vs. Fab-bound peptide were found to be largest for the amide groups of Asp-1 and Asp-6, in agreement with classification of these residues as critical by the phage display library selection process. For peptide 2 the largest absolute shifts were observed for Asp-1 and Asp-4, with the other aspartic acid residues also showing significant but smaller changes.
Stockman
B J
BJ
Upjohn Laboratories, Upjohn Company, Kalamazoo, Michigan, USA.
Bannow
C A
CA
Miceli
R M
RM
Degraaf
M E
ME
Fischer
H D
HD
Smith
C W
CW
D000595:Amino Acid Sequence; D000937:Antigen-Antibody Reactions; D001435:Bacteriophages / Q000235:genetics*; D015698:Genomic Library; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry*; D008969:Molecular Sequence Data; D010455:Peptides / Q000276:immunology*; D011897:Random Allocation
D007140:Immunoglobulin Fab Fragments; D010455:Peptides