Histidine Hydrogen-Deuterium Exchange Mass Spectrometry for Probing the Microenvironment of Histidine Residues in Dihydrofolate Reductase

Background Histidine Hydrogen-Deuterium Exchange Mass Spectrometry (His-HDX-MS) determines the HDX rates at the imidazole C 2 -hydrogen of histidine residues. This method provides not only the HDX rates but also the p K a values of histidine imidazole rings. His-HDX-MS was used to probe the microenvironment of histidine residues of E. coli dihydrofolate reductase (DHFR), an enzyme proposed to undergo multiple conformational changes during catalysis. Methodology/Principal Findings Using His-HDX-MS, the p K a values and the half-lives ( t 1/2 ) of HDX reactions of five histidine residues of apo - DHFR, DHFR in complex with methotrexate (DHFR-MTX), DHFR in complex with MTX and NADPH (DHFR-MTX-NADPH), and DHFR in complex with folate and NADP + (DHFR-folate-NADP + ) were determined. The results showed that the two parameters (p K a and t 1/2 ) are sensitive to the changes of the microenvironment around the histidine residues. Although four of the five histidine residues are located far from the active site, ligand binding affected their p K a , t 1/2 or both. This is consistent with previous observations of ligand binding-induced distal conformational changes on DHFR. Most of the observed p K a and t 1/2 changes could be rationalized using the X-ray structures of apo-DHFR, DHFR-MTX-NADPH, and DHFR-folate-NADP + . The availability of the neutron diffraction structure of DHFR-MTX enabled us to compare the protonation states of histidine imidazole rings. Conclusions/Significance Our results demonstrate the usefulness of His-HDX-MS in probing the microenvironments of histidine residues within proteins.