PMID:12660154
| Citation |
Weinglass, AB, Whitelegge, JP, Hu, Y, Verner, GE, Faull, KF and Kaback, HR (2003) Elucidation of substrate binding interactions in a membrane transport protein by mass spectrometry. EMBO J. 22:1467-77 |
|---|---|
| Abstract |
Integration of biochemical and biophysical data on the lactose permease of Escherichia coli has culminated in a molecular model that predicts substrate-protein proximities which include interaction of a hydroxyl group in the galactopyranosyl ring with Glu269. In order to test this hypothesis, we studied covalent modification of carboxyl groups with carbodiimides using electrospray ionization mass spectrometry (ESI-MS) and demonstrate that substrate protects the permease against carbodiimide reactivity. Further more, a significant proportion of the decrease in carbodiimide reactivity occurs specifically in a nanopeptide containing Glu269. In contrast, carbodiimide reactivity of mutant Glu269-->Asp that exhibits lower affinity is unaffected by substrate. By monitoring the ability of different substrate analogs to protect against carbodiimide modification of Glu269, it is suggested that the C-3 OH group of the galactopyranosyl ring may play an important role in specificity, possibly by H-bonding with Glu269. The approach demonstrates that mass spectrometry can provide a powerful means of analyzing ligand interactions with integral membrane proteins. |
| Links |
PubMed PMC152890 Online version:10.1093/emboj/cdg145 |
| Keywords |
Amino Acid Substitution; Carbodiimides/chemistry; Chromatography, High Pressure Liquid; Escherichia coli/enzymology; Escherichia coli Proteins; Membrane Transport Proteins/chemistry; Membrane Transport Proteins/metabolism; Monosaccharide Transport Proteins; Peptide Fragments/chemistry; Peptide Fragments/metabolism; Protein Binding; Protein Conformation; Spectrometry, Mass, Electrospray Ionization; Substrate Specificity; Symporters |
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