Defining topological features of membrane proteins by nanoelectrospray ionisation mass spectrometry.
The D-galactose-H(+) symport protein, GalP, of Escherichia coli is the bacterial homologue of the human glucose transport protein, GLUT1. Here we demonstrate that mass spectrometry can be used to map modification by covalently bound reagents, and also to detect structural changes in the GalP protein that occur upon substrate binding. The small thiol-group-specific reagent N-ethylmaleimide (NEM) was used to modify the cysteine residues in GalP(His)(6) both alone and in the presence of D-glucose, a known substrate. Employing a mixture of proteolysis and thermal degradation methods, the three cysteine residues were found to undergo sequential reactions with NEM, with Cys374 being modified first, followed by Cys389 and finally Cys19, thus indicating their different accessibilities within the three-dimensional structure of the protein. Prior binding of the substrate D-glucose to the protein protected Cys19 and Cys374 against NEM modification, but not Cys389. Cys374 had been expected to be shielded by D-glucose binding while Cys389 had been expected to be unaffected, consistent with their proposed respective locations in the vicinity of, and distant from, the sugar binding site. However, the inaccessibility of Cys19 was unexpected and suggests a structural change in the protein promoted by D-glucose binding which changes the proximity of Cys19 with respect to the D-glucose-binding site.
Jones
Lynsey N
LN
Astbury Centre for Structural Molecular Biology, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, UK.
Baldwin
Stephen A
SA
Henderson
Peter J F
PJ
Ashcroft
Alison E
AE
D002135:Calcium-Binding Proteins / Q000032:analysis / Q000378:metabolism*; D004926:Escherichia coli / Q000166:cytology / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000032:analysis / Q000378:metabolism*; D005947:Glucose / Q000378:metabolism; D008958:Models, Molecular; D009004:Monosaccharide Transport Proteins / Q000032:analysis / Q000378:metabolism*; D033902:Periplasmic Binding Proteins / Q000032:analysis / Q000378:metabolism*; D011485:Protein Binding; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
D002135:Calcium-Binding Proteins; D029968:Escherichia coli Proteins; D009004:Monosaccharide Transport Proteins; D033902:Periplasmic Binding Proteins; C023550:galactose-binding protein; D005947:Glucose