Abstract
The mini-protein T4 Fibritin foldon (T4Ff) allows targeted protein-protein interaction studies by protein engineering which alters primary structure to influence higher-order structures. T4Ff variants revealed how non-natural N-terminally located amino acid residues with large π-electron rings affected trimer stability. Mass spectrometric ITEM-FIVE analyses determined apparent kinetic and quasi-thermodynamic properties of trimer dissociation reactions in the gas phase. Results presented here show that aromatic π-stacking stabilizes T4Ff variant homo-trimers which otherwise were less stable than the original T4Ff because of inner-chain amino acid exchanges. The T4Ff activation enthalpy of 63.9 kJ/mol dropped to 51.8 kJ/mol for its Gly10→D-Ala and Asp17→D-Phe-containing variant. This drop was counterbalanced by additionally placing amino acid residues with aromatic side chains at the N-termini. The diphenyl amino acid side chain-carrying variant is slightly more stable than the original T4Ff (activation enthalpy: 66.8 kJ/mol). ITEM-FIVE proved capable of determining non-covalent force differences with amino acid residue resolution.