Journal
BIOCHEMISTRY
Volume 45, Issue 51, Pages 15279-15287Publisher
AMER CHEMICAL SOC
DOI: 10.1021/bi061632m
Keywords
-
Categories
Funding
- NIDDK NIH HHS [R01 DK051131-13, R01 DK069463, DK069463, R13 DK058527-01, R56 DK051131, DK051131, R01 DK051131, R01 DK069463-04, R56 DK069463] Funding Source: Medline
- NIGMS NIH HHS [U54 GM074929, U54 GM074929-040005] Funding Source: Medline
Ask authors/readers for more resources
Trp151 in the lactose permease of Escherichia coli (LacY) is an important component of the sugar-binding site and the only Trp residue out of six that is in close proximity to the galactopyranoside in the structure (1PV7). The short distance between Trp151 and the sugar is favorable for Forster resonance energy transfer (FRET) to nitrophenyl or dansyl derivatives with the fluorophore at the anomeric position of galactose. Modeling of 4-nitrophenyl-alpha-D-galactopyranoside (alpha-NPG) in the binding-site of LacY places the nitrophenyl moiety about 12 A away from Trp151, a distance commensurate with the Forster distance for a Trp-nitrobenzoyl pair. We demonstrate here that alpha-NPG binding to LacY containing all six native Trp residues causes galactopyranoside-specific FRET from Trp151. Moreover, binding of alpha-NPG is sufficiently slow to resolve time-dependent fluorescence changes by stopped-flow. The rate of change in Trp -> alpha-NPG FRET is linearly dependent upon sugar concentration, which allows estimation of kinetic parameters for binding. Furthermore, 2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid (MIANS) covalently attached to the cytoplasmic end of helix X is sensitive to sugar binding, reflecting a ligand-induced conformational change. Stopped-flow kinetics of Trp -> alpha-NPG FRET and sugar-induced changes in MIANS fluorescence in the same protein reveal a two-step process: a relatively rapid binding step detected by Trp151 -> alpha-NPG FRET followed by a slower conformational change detected by a change in MIANS fluorescence.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available