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Elucidation of substrate binding interactions in a membrane transport protein by mass spectrometry
Author(s) -
Weinglass Adam B.,
Whitelegge Julian P.,
Hu Yonglin,
Verner Gillian E.,
Faull Kym F.,
Kaback H. Ronald
Publication year - 2003
Publication title -
the embo journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 7.484
H-Index - 392
eISSN - 1460-2075
pISSN - 0261-4189
DOI - 10.1093/emboj/cdg145
Subject(s) - biology , mass spectrometry , membrane protein , plasma protein binding , biophysics , substrate (aquarium) , transport protein , membrane , membrane transport protein , proteomics , substrate specificity , biochemistry , binding site , microbiology and biotechnology , chromatography , enzyme , chemistry , ecology , gene
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.