Elucidation of substrate binding interactions in a membrane transport protein by mass spectrometry.
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.
Weinglass
Adam B
AB
Department of Physiology, Molecular Biology Institute, University of California Los Angeles, Los Angeles, CA 90095-1662, USA.
Whitelegge
Julian P
JP
Hu
Yonglin
Y
Verner
Gillian E
GE
Faull
Kym F
KF
Kaback
H Ronald
HR
D019943:Amino Acid Substitution; D002234:Carbodiimides / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000378:metabolism*; D009004:Monosaccharide Transport Proteins; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D021241:Spectrometry, Mass, Electrospray Ionization; D013379:Substrate Specificity; D027981:Symporters
D002234:Carbodiimides; D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D010446:Peptide Fragments; D027981:Symporters; C021499:lactose permease