title: Probing the influence of mutations on the stability of a ferredoxin by mass spectrometry.
abstract: Hydrogen/deuterium exchange, which depends on solvent accessibility, can be probed by mass spectrometry (MS) to get information on protein conformation or protein-ligand interaction. In this work, the conformational properties of the cyanobacterium Anabaena wild-type ferredoxin as well as of two single-site mutants (Phe 65 Ala and Arg 42 Ala) were studied. After incubation of the wild type and mutant proteins in deuterated water and quenching of the exchange at low pH, the proteins were rapidly digested at high enzyme-to-substrate ratio using immobilized pepsin, and the resulting peptides were characterized using ESI-MS. We have identified specific regions for which the H-bonding or solvent accessibility properties were perturbed by the mutations. These results show that this approach can provide local information on the influence of mutations, even for a highly structured protein like ferredoxin, and sometimes in regions distant from the mutation point.
authors.affiliation: Institut de Biologie Structurale, Grenoble, France.
authors.forename: H
authors.initials: H
authors.lastname: Rémigy
authors.forename: M
authors.initials: M
authors.lastname: Jaquinod
authors.forename: Y
authors.initials: Y
authors.lastname: Pétillot
authors.forename: J
authors.initials: J
authors.lastname: Gagnon
authors.forename: H
authors.initials: H
authors.lastname: Cheng
authors.forename: B
authors.initials: B
authors.lastname: Xia
authors.forename: J L
authors.initials: JL
authors.lastname: Markley
authors.forename: J K
authors.initials: JK
authors.lastname: Hurley
authors.forename: G
authors.initials: G
authors.lastname: Tollin
authors.forename: E
authors.initials: E
authors.lastname: Forest
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics; D000458:Cyanobacteria / Q000235:genetics / Q000378:metabolism; D003903:Deuterium; D004355:Drug Stability; D005288:Ferredoxins / Q000737:chemistry* / Q000235:genetics*; D013058:Mass Spectrometry; D009154:Mutation; D011487:Protein Conformation
chemical_list: D001426:Bacterial Proteins; D005288:Ferredoxins; D003903:Deuterium