Site-resolved energetic information from HX/MS experiments

High-resolution energetic information about protein conformational ensembles is essential for understanding protein function, yet remains challenging to obtain. We present PIGEON-FEATHER, a method for calculating ensemble free energies of opening (ΔG_op) at single- or near-single-amino acid resolution for proteins of all sizes from hydrogen exchange/mass spectrometry (HX/MS) data. PIGEON-FEATHER disambiguates and reconstructs all experimentally measured HX/MS isotopic mass envelopes using a Bayesian Monte Carlo sampling approach. We applied PIGEON-FEATHER to reveal how E. coli and human dihydrofolate reductases (ecDHFR, hDHFR) have evolved distinct ensembles. We show how two competitive inhibitors bind these orthologs differently, solving the longstanding mystery of why both therapeutic molecules inhibit hDHFR, but only one inhibits ecDHFR. Extending PIGEON-FEATHER to a large protein-DNA complex, we mapped ligand-induced ensemble reweighting in the E. coli lac repressor to describe the functional switching mechanism crucial for transcriptional regulation.