Site-resolved energetic information from HX/MS experiments

Abstract

While bioinformatics reveals patterns in protein sequences and structural biology methods elucidate atomic details of protein structures, it is difficult to attain equally high-resolution energetic information about protein conformational ensembles. We present PIGEON-FEATHER, a method for calculating free energies of opening (∆Gop) at single- or near-single-amino acid resolution for protein ensembles of all sizes from hydrogen exchange/mass spectrometry (HX/MS) data. PIGEON-FEATHER disambiguates and reconstructs all experimentally measured isotopic mass envelopes using a Bayesian Monte Carlo sampling approach. We applied PIGEON-FEATHER to reveal how E. coli and human dihydrofolate reductase orthologs (ecDHFR, hDHFR) have evolved distinct ensembles tuned to their catalytic cycles, and how two competitive inhibitors of ecDHFR arrest its ensemble in different ways. Extending the method to a large protein-DNA complex, we mapped ligand-induced ensemble reweighting in the E. coli lac repressor to understand the functional switching mechanism crucial for transcriptional regulation.