|
 |
|
|
| Subject Categories: Membranes & Transport | Microbiology & Pathogens |
|
| The EMBO Journal
(2003) 22, 1467–1477, doi:10.1093/emboj/cdg145 |
|
| Elucidation of substrate binding interactions in a membrane transport protein by mass spectrometry |
|
|
| Adam B. Weinglass1, Julian P. Whitelegge2, Yonglin Hu1, Gillian E. Verner1, Kym F. Faull2 and H. Ronald Kaback1 |
|
|
1 Howard Hughes Medical Institute, Departments of Physiology and Microbiology and Molecular Genetics, Molecular Biology Institute, University of California Los Angeles, Los Angeles, CA 90095-1662, USA 2 The Pasarow Mass Spectrometry Laboratory, Departments of Psychiatry and Behavioural Sciences, The Neuropsychiatric Institute, and The Department of Chemistry and Biochemistry, University of California Los Angeles, Los Angeles, CA 90095-1662, USA To whom correspondence should be addressed H. Ronald Kaback, RonaldK@HHMI.UCLA.edu Received 21 November 2002; Revised 30 January 2003; Accepted 4 February 2003. |
|
|
|
| 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. |
|
| Keywords: bioenergetics, H+ symport, lactose permease, ligand binding, membrane proteins |
|
|
| |
| |
| |
 | Email link to a friend |
| |
 | Download PDF |
| |
 | Full text |
| |
| |
| |
 | Next article |
| |
 | Previous article |
| |
 | Table of contents |
| |
| |
 | Rights and permissions |
| |
 | Reprints |
| |
| |
| |
 Register for table of contents by email | |
| |
| |
| |