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

Abstract

BackgroundHistidine Hydrogen-Deuterium Exchange Mass Spectrometry (His-HDX-MS) determines the HDX rates at the imidazole C2-hydrogen of histidine residues. This method provides not only the HDX rates but also the pK 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 FindingsUsing His-HDX-MS, the pK 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 (pK 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 pK 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 pK 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/SignificanceOur results demonstrate the usefulness of His-HDX-MS in probing the microenvironments of histidine residues within proteins.

Conformational Stability of Mammalian Prion Protein Amyloid Fibrils Is Dictated by a Packing Polymorphism within the Core Region

“…His-HXMS-Structural differences between the two fibril types were further probed using the recently developed method of His-HXMS (30,31,39). In this approach, the rate of H/D exchange for C2 protons in histidine imidazole rings is measured.…”

“…Even for unprotected histidines, this exchange is relatively slow (on the order of ϳ2 days), allowing convenient monitoring by mass spectrometry. If the His side chain is buried in a water-protected environment, this exchange time is increased to many weeks or even months (30,31). Amide HXMS and His-HXMS methods are highly complementary: whereas the former probes protein structure at the level of the polypeptide backbone, the latter provides information regarding the microenvironment (water accessibility) of specific His side chains.…”

Quantitative Measurement of the Solvent Accessibility of Histidine Imidazole Groups in Proteins

“…However, mass spectrometry (MS) is gaining popularity in recent years, because MS provides higher sensitivity and straightforward signal assignment, and is capable of analyzing membrane proteins and complex protein mixtures (7, 9–12). The MS method has been referred to as histidine hydrogen-deuterium exchange mass spectrometry (His-HDX-MS) (9–11) in order to distinguish it from the amide-HDX-MS (13). …”

Determination of Histidine pKa Values in the Propeptides of Furin and Proprotein Convertase 1/3 Using Histidine Hydrogen–Deuterium Exchange Mass Spectrometry

“…24 However, scrambling during CID appears not to affect deuterium bound to the C2 of the imidazole side chain. 25,26 The CID-MS2 spectrum identifies fragment y47 (+8 charged state) as the most intense ion (Figure 1D). The OrbiTrap Fusion allows the selection of the y47 ion for subsequent MS3 fragmentation using CID, which yields the fragment y47_b6, that only which contains only His 66 .…”