title: Comparative hydrogen-deuterium exchange for a mesophilic vs thermophilic dihydrofolate reductase at 25 °C: identification of a single active site region with enhanced flexibility in the mesophilic protein.
abstract: The technique of hydrogen-deuterium exchange coupled to mass spectrometry (HDX-MS) has been applied to a mesophilic (E. coli) dihydrofolate reductase under conditions that allow direct comparison to a thermophilic (B. stearothermophilus) ortholog, Ec-DHFR and Bs-DHFR, respectively. The analysis of hydrogen-deuterium exchange patterns within proteolytically derived peptides allows spatial resolution, while requiring a series of controls to compare orthologous proteins with only ca. 40% sequence identity. These controls include the determination of primary structure effects on intrinsic rate constants for HDX as well as the use of existing 3-dimensional structures to evaluate the distance of each backbone amide hydrogen to the protein surface. Only a single peptide from the Ec-DHFR is found to be substantially more flexible than the Bs-DHFR at 25 °C in a region located within the protein interior at the intersection of the cofactor and substrate-binding sites. The surrounding regions of the enzyme are either unchanged or more flexible in the thermophilic DHFR from B. stearothermophilus. The region with increased flexibility in Ec-DHFR corresponds to one of two regions previously proposed to control the enthalpic barrier for hydride transfer in Bs-DHFR [Oyeyemi et al. (2010) Proc. Natl. Acad. Sci. U.S.A. 107, 10074].
authors.affiliation: Department of Chemistry, California Institute for Quantitative Biosciences (QB3), University of California, Berkeley, Berkeley, California 94720, USA.
authors.forename: Olayinka A
authors.initials: OA
authors.lastname: Oyeyemi
authors.forename: Kevin M
authors.initials: KM
authors.lastname: Sours
authors.forename: Thomas
authors.initials: T
authors.lastname: Lee
authors.forename: Amnon
authors.initials: A
authors.lastname: Kohen
authors.forename: Katheryn A
authors.initials: KA
authors.lastname: Resing
authors.forename: Natalie G
authors.initials: NG
authors.lastname: Ahn
authors.forename: Judith P
authors.initials: JP
authors.lastname: Klinman
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020134:Catalytic Domain; D041961:Deuterium Exchange Measurement; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001411:Geobacillus stearothermophilus / Q000201:enzymology / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D040681:Structural Homology, Protein; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013816:Thermodynamics
chemical_list: D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D013762:Tetrahydrofolate Dehydrogenase