Binding of spin-labeled galactosides to the lactose permease of Escherichia coli

Citation
M. Zhao et al., Binding of spin-labeled galactosides to the lactose permease of Escherichia coli, BIOCHEM, 39(37), 2000, pp. 11381-11388
Citations number
39
Categorie Soggetti
Biochemistry & Biophysics
Journal title
BIOCHEMISTRY
ISSN journal
00062960 → ACNP
Volume
39
Issue
37
Year of publication
2000
Pages
11381 - 11388
Database
ISI
SICI code
0006-2960(20000919)39:37<11381:BOSGTT>2.0.ZU;2-E
Abstract
A series of nitroxide spin-labeled alpha- or beta-galactopyranosides and a nitroxide spin-labeled beta-glucopyranoside have been synthesized and exami ned for binding to the lactose permease of Escherichia coli. Out of the twe lve nitroxide spin-labeled galactopyranosides synthesized, 1-oxyl-2, 5, 5-t rimethyl-2-[3-nitro-4-N-(hexyl-1-thio-beta-D-galactopyranosid-1-yl)]aminoph enyl pyrrolidine (NNG(6)(beta)) exhibits the highest affinity for the perme ase based on the following observations: (a) the analogue inhibits lactose transport with a K-I about 7 mu M; (b) NNG(6)(beta) blocks labeling of sing le-Cys148 permease with 2-(4'-maleimidylanilino) naphthalene-6-sulfonic aci d (MIANS) with an apparent affinity of about 12 mu M; (c) electron paramagn etic resonance demonstrates binding of the spin-labeled sugar by purified w ild-type permease in a manner that is reversed by nonspin-labeled ligand, T he equilibrium dissociation constant (K-D) is about 23 mu M and binding sto ichiometry is approximately unity. In contrast, the nitroxide spin-labeled glucopyranoside does not inhibit active lactose transport or labeling of si ngle-Cys148 permease with MIANS, It is concluded that NNG(6)(beta) binds sp ecifically to lac permease with an affinity in the low micromolar range. Fu rthermore, affinity of the permease for the spin-labeled galactopyranosides is directly related to the length, hydrophobicity, and geometry of the lin ker between the galactoside and the nitroxide spin-label.