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 (NN) exhibits the highest affinity for the permease based on the following observations: (a) the analogue inhibits lactose transport with a K(I) about 7 microM; (b) NN blocks labeling of single-Cys148 permease with 2-(4'-maleimidylanilino) naphthalene-6-sulfonic acid (MIANS) with an apparent affinity of about 12 microM; (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 microM 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 NN 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.
Zhao
M
M
Molecular Biology Institute, Departments of Physiology and Microbiology & Molecular Genetics, Howard Hughes Medical Institute; Institute of Organic and Medicinal Chemistry, University of Pécs, H-7643 Pécs, P.O. Box 99, Hungary.
Kálai
T
T
Hideg
K
K
Altenbach
C
C
Hubbell
W L
WL
Kaback
H R
HR
D000477:Alkylating Agents / Q000378:metabolism; D000817:Anilino Naphthalenesulfonates / Q000378:metabolism; D001665:Binding Sites; D001692:Biological Transport; D003545:Cysteine / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D005690:Galactose / Q000378:metabolism; D007785:Lactose / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000378:metabolism*; D009004:Monosaccharide Transport Proteins; D017433:Protein Structure, Secondary; D011759:Pyrrolidines / Q000737:chemistry / Q000378:metabolism*; D013113:Spin Labels; D019097:Spin Trapping; D027981:Symporters; D013862:Thiogalactosides / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*
C415245:1-oxyl-2,5,5-trimethyl-2-(3-nitro-4-N-(hexyl-1-thiogalactopyranosid-1-yl))aminophenylpyrrolidine; D000477:Alkylating Agents; D000817:Anilino Naphthalenesulfonates; D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D011759:Pyrrolidines; D013113:Spin Labels; D027981:Symporters; D013862:Thiogalactosides; C021310:2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid; C021499:lactose permease; D007785:Lactose; D003545:Cysteine; D005690:Galactose