title: Histidine hydrogen-deuterium exchange mass spectrometry for probing the microenvironment of histidine residues in dihydrofolate reductase.
abstract: 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 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.
  
  
  RESULTS
  Using 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
  Our results demonstrate the usefulness of His-HDX-MS in probing the microenvironments of histidine residues within proteins.
authors.affiliation: Case Center for Proteomics and Bioinformatics, Case Western Reserve University, Cleveland, Ohio, United States of America. masaru.miyagi@case.edu
authors.forename: Masaru
authors.initials: M
authors.lastname: Miyagi
authors.forename: Qun
authors.initials: Q
authors.lastname: Wan
authors.forename: Md Faiz
authors.initials: MF
authors.lastname: Ahmad
authors.forename: Giridharan
authors.initials: G
authors.lastname: Gokulrangan
authors.forename: Sara E
authors.initials: SE
authors.lastname: Tomechko
authors.forename: Brad
authors.initials: B
authors.lastname: Bennett
authors.forename: Chris
authors.initials: C
authors.lastname: Dealwis
mesh_terms: D020134:Catalytic Domain; D003461:Crystallography / Q000379:methods; D003903:Deuterium / Q000737:chemistry / Q000378:metabolism; D041961:Deuterium Exchange Measurement / Q000379:methods*; D004777:Environment; D004926:Escherichia coli / Q000737:chemistry / Q000201:enzymology; D006639:Histidine / Q000737:chemistry* / Q000378:metabolism; D006859:Hydrogen / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D013058:Mass Spectrometry / Q000379:methods*; D008954:Models, Biological; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D011485:Protein Binding; D011487:Protein Conformation; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry* / Q000378:metabolism
chemical_list: D006639:Histidine; D006859:Hydrogen; D003903:Deuterium; D013762:Tetrahydrofolate Dehydrogenase