The mass spectrometric intact transition epitope mapping method supports protein engineering of foldon trimer variants

The wild-type foldon (foldon 0) is the C-terminal trimerization domain of the bacteriophage T4 Fibritin foldon (T4Ff). T4Ff’s amino acid sequence is GYIPEAPRDGQAYVRKDGEWVLLSTFL (27 amino acid residues; single letter code). Foldons 0, 1-6 were synthesized by solid phase peptide synthesis and in foldons 1-6 D amino acids at positions 10 (G10a) and 17 (D17f) were introduced. In addition, foldons 2-6 carry artificial N-termini consisting of amino acid residues with increasing space-demanding side chains. All foldons, 0-6, form non-covalent trimers in solution and courses of trimer dissociations were investigated after electrospraying supramolecular ions in the gas phase using ITEM-FIVE (Intact Transition Epitope Mapping - Force Interferences by Variable Extensions) mass spectrometry. Differences in trimer stabilities were correlated with structural features.