PMID:15272008
| Citation |
Weinglass, A, Whitelegge, JP, Faull, KF and Kaback, HR (2004) Monitoring conformational rearrangements in the substrate-binding site of a membrane transport protein by mass spectrometry. J. Biol. Chem. 279:41858-65 |
|---|---|
| Abstract |
Combined biochemical, biophysical, and crystallographic studies on the lactose permease of Escherichia coli suggest that Arg-144 (helix V) forms a salt bridge with Glu-126 (helix IV), which is broken during substrate binding, thereby permitting the guanidino group to form a bidentate H-bond with the C-4 and C-3 O atoms of the galactopyranosyl moiety and an H-bond with Glu-269 (helix VIII). To examine the relative interaction of Arg-144 with these two potential salt bridge partners (Glu-126 and Glu-269) in the absence of substrate, the covalent modification of the guanidino group was monitored with the Arg-specific reagent butane-2,3-dione using electrospray ionization mass spectrometry. In a functional background, the reactivity of Arg-144 with butane-2,3-dione is low ( approximately 25%) and is reduced by a factor of approximately 2 by preincubation with ligand. Interestingly, although replacement of Glu-126 with Ala results in a 3-fold increase in the reactivity of Arg-144, replacement of Glu-269 with Ala elicits virtually no effect. Taken together, these results suggest that in the absence of substrate the interaction between Arg-144 and Glu-126 is much stronger than the interaction with Glu-269, supporting the contention that sugar recognition leads to rearrangement of charge-paired residues essential for sugar binding. |
| Links |
PubMed Online version:10.1074/jbc.M407555200 |
| Keywords |
Arginine; Binding Sites; Escherichia coli Proteins/chemistry; Glutamic Acid; Membrane Transport Proteins/chemistry; Monosaccharide Transport Proteins/chemistry; Protein Conformation; Spectrometry, Mass, Electrospray Ionization/methods; Static Electricity; Symporters/chemistry |
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