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

A series of nitroxide spin-labeled alpha- or beta-galactopyranosides and a nitroxide spin-labeled beta-glucopyranoside have been synthesized and examined for binding to the lactose permease of Escherichia coli. Out of the twelve nitroxide spin-labeled galactopyranosides synthesized, 1-oxyl-2, 5, 5-trimethyl-2-[3-nitro-4-N-(hexyl-1-thio-beta-D-galactopyranosid-1-yl)]aminophenyl pyrrolidine (NNG(6)(beta)) exhibits the highest affinity for the permease 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 single-Cys148 permease with 2-(4'-maleimidylanilino) naphthalene-6-sulfonic acid (MIANS) with an apparent affinity of about 12 mu M; (c) electron paramagnetic resonance demonstrates binding of the spin-labeled sugar by purified wild-type permease in a manner that is reversed by nonspin-labeled ligand, The equilibrium dissociation constant (K-D) is about 23 mu M and binding stoichiometry is approximately unity. In contrast, the nitroxide spin-labeled glucopyranoside does not inhibit active lactose transport or labeling of single-Cys148 permease with MIANS, It is concluded that NNG(6)(beta) binds specifically to lac permease with an affinity in the low micromolar range. Furthermore, affinity of the permease for the spin-labeled galactopyranosides is directly related to the length, hydrophobicity, and geometry of the linker between the galactoside and the nitroxide spin-label.