Rational design of a foldon-derived heterotrimer guided by quantitative native mass spectrometry

Designing stable hetero-oligomeric protein complexes with defined inter-subunit stoichiometries remains a significant challenge. In this study, we report the design of a highly selective heterotrimeric assembly derived from the well-known foldon homotrimer. We generated an aab heterotrimer by introducing the Q11E modification to destabilize the homotrimer and a compensatory modification, either V14A or V14L, to stabilize the hydrophobic core of the heterotrimer. Native mass spectrometry (MS) was essential for guiding the design process, enabling precise characterization of oligomeric states and their equilibrium distributions. The heterotrimer structure was validated by x-ray crystallography. Our findings highlight the effectiveness of combining rational design with native MS to develop specific hetero-oligomeric assemblies.