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

## TL;DR

Researchers used a mass spectrometry method to study how amino acid changes affect the stability of a mini-protein called T4Ff.

## Contribution

A new mass spectrometric method (ITEM-FIVE) was used to analyze trimer stability with amino acid resolution.

## Key findings

- Aromatic π-stacking at N-termini stabilizes T4Ff variants despite destabilizing inner-chain exchanges.
- A diphenyl amino acid variant showed higher stability than original T4Ff (activation enthalpy: 66.8 kJ/mol).
- ITEM-FIVE can detect non-covalent force differences at the amino acid level in gas-phase trimers.

## 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.

The online version contains supplementary material available at 10.1038/s41598-025-28101-7.