2025 Theses Doctoral

Quantitative high-resolution HX-MS to discover protein energetic landscapes

Wells, Malcolm

Structural biologists use hydrogen exchange mass spectrometry (HX-MS) to study freeenergies of local unfolding in proteins, typically at medium sequence resolution and by qualitative comparisons between functional states (e.g., wild-type proteins vs. mutants, apo proteins vs ligand-bound). HX-MS reports in principle on the free energies of opening of individual residues, but typical experiments are interpreted with much less structural detail and consequently no ability to calculate free energies. We developed PIGEON-FEATHER, a computational workflow for HX-MS that accomplishes superior coverage and accuracy in peptide identification in the HX-MS context, and fits free energies of opening at near amino-acid resolution, allowing detailed and quantitative analysis of local stability in proteins. This work is described in Chapter 2.

We then applied PIGEON-FEATHER to several interesting protein systems. First, we discovered distinct energetic profiles in human and E. coli DHFR, revealing how different organisms adapted the same fold to the demands of catalysis in different environments, and furthermore identified distinct mechanisms of action for the antibiotic trimethoprim and the broad-spectrum DHFR inhibitor methotrexate (also described in Chapter 2).

Next, as described in chapter 3, we collected one of the largest HX-MS datasets to date and applied HX-MS/PIGEON-FEATHER in conjunction with crystallographic, bioinformatic, and mutational/functional methods to discover an evolutionarily conserved set of coupled energetic and structural changes underlying function in the Venus flytrap (VFT)-fold LacI/GalR family of transcription factors. We studied in particular E. coli lac repressor (LacI), ribose repressor (RbsR), and galactose repressor (GalR).

In addition, we compared the evolutionarily diverged monomeric periplasmic binding proteins RbsB and MglB to reveal how the same ancient VFT fold adopts distinct energetic responses to binding the same ligands, for a different biological function. Measuring HX at residue resolution enabled us to understand in detail how functional effects of ligand binding are conserved and diversified in the VFT fold. Quantitative HX-MS can reveal the molecular basis for disease and offer new paths to therapeutic interventions. We engaged in two collaborations on cancer- and viral infection-relevant protein systems, described in Chapter 4.

First, we used PIGEON-enabled HX-MS to show that the Src homology region 2-containing protein tyrosine phosphatase 2 (SHP2) hotspot phosphorylation pY62, implicated in cancer, achieves oncogenesis and inhibitor resistance by destabilizing the closed, autoinhibited conformation in favor of the open, active one. Next, we compared the postfusion and prefusion-stabilized states of the herpes simplex virus 1 (HSV-1) glycoprotein B (gB) using HX-MS and found that gB is energetically remodeled at domain interfaces by the dramatic conformational changes it undergoes, despite high structural similarity within domains; we further identified bimodal exchange behavior in several refolding regions indicating the presence of multiple slowly-interconverting conformations in these regions. Altogether, this work demonstrates the power of accurate, near amino acid-resolved HX-MS for understanding how protein ensembles, beyond the ground state structure, impact their function.