title: Molecular dissection of membrane-transport proteins: mass spectrometry and sequence determination of the galactose-H+ symport protein, GalP, of Escherichia coli and quantitative assay of the incorporation of [ring-2-13C]histidine and (15)NH(3).
abstract: The molecular mass of the galactose-H(+) symport protein GalP, as its histidine-tagged derivative GalP(His)(6), has been determined by electrospray MS (ESI-MS) with an error of <0.02%. One methionine residue, predicted to be present from the DNA sequence, was deduced to be absent. This is a significant advance on the estimation of the molecular masses of membrane-transport proteins by SDS/PAGE, where there is a consistent under-estimation of the true molecular mass due to anomalous electrophoretic migration. Addition of a size-exclusion chromatography step after Ni(2+)-nitrilotriacetate affinity purification was essential to obtain GalP(His)(6) suitable for ESI-MS. Controlled trypsin, trypsin+chymotrypsin and CNBr digestion of the protein yielded peptide fragments suitable for ESI-MS and tandem MS analysis, and accurate mass determination of the derived fragments resulted in identification of 82% of the GalP(His)(6) protein. Tandem MS analysis of selected peptides then afforded 49% of the actual amino acid sequence of the protein; the absence of the N-terminal methionine was confirmed. Matrix-assisted laser-desorption ionization MS allowed identification of one peptide that was not detected by ESI-MS. All the protein/peptide mass and sequence determinations were in accord with the predictions of amino acid sequence deduced from the DNA sequence of the galP gene. [ring-2-(13)C]Histidine was incorporated into GalP(His)(6) in vivo, and ESI-MS analysis enabled the measurement of a high (80%) and specific incorporation of label into the histidine residues in the protein. MS could also be used to confirm the labelling of the protein by (15)NH(3) (93% enrichment) and [(19)F]tryptophan (83% enrichment). Such MS measurements will serve in the future analysis of the structures of membrane-transport proteins by NMR, and of their topology by indirect techniques.
authors.affiliation: Astbury Centre for Structural Molecular Biology, School of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9JT, U.K.
authors.forename: Henrietta
authors.initials: H
authors.lastname: Venter
authors.forename: Alison E
authors.initials: AE
authors.lastname: Ashcroft
authors.forename: Jeffrey N
authors.initials: JN
authors.lastname: Keen
authors.forename: Peter J F
authors.initials: PJ
authors.lastname: Henderson
authors.forename: Richard B
authors.initials: RB
authors.lastname: Herbert
mesh_terms: D000595:Amino Acid Sequence; D000641:Ammonia / Q000378:metabolism; D002135:Calcium-Binding Proteins; D002247:Carbon Isotopes; D003488:Cyanogen Bromide; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005690:Galactose / Q000378:metabolism; D006639:Histidine / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009004:Monosaccharide Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D009587:Nitrogen Isotopes; D010446:Peptide Fragments / Q000302:isolation & purification; D033902:Periplasmic Binding Proteins; D021241:Spectrometry, Mass, Electrospray Ionization
chemical_list: D002135:Calcium-Binding Proteins; D002247:Carbon Isotopes; D029968:Escherichia coli Proteins; D009004:Monosaccharide Transport Proteins; D009587:Nitrogen Isotopes; D010446:Peptide Fragments; D033902:Periplasmic Binding Proteins; C023550:galactose-binding protein; D006639:Histidine; D000641:Ammonia; D003488:Cyanogen Bromide; D005690:Galactose