Nitroxide scanning electron paramagnetic resonance of helices IV and V andthe intervening loop in the lactose permease of Escherichia coli

Citation
M. Zhao et al., Nitroxide scanning electron paramagnetic resonance of helices IV and V andthe intervening loop in the lactose permease of Escherichia coli, BIOCHEM, 38(48), 1999, pp. 15970-15977
Citations number
45
Categorie Soggetti
Biochemistry & Biophysics
Journal title
BIOCHEMISTRY
ISSN journal
00062960 → ACNP
Volume
38
Issue
48
Year of publication
1999
Pages
15970 - 15977
Database
ISI
SICI code
0006-2960(19991130)38:48<15970:NSEPRO>2.0.ZU;2-J
Abstract
Glu126 and Arg144 in helices TV and V, respectively, in the lactose permeas e of Escherichia coli, which play an indispensable role in substrate bindin g, are charge-paired and in close proximity [Venkatesan, P., Kaback, I-I. R . (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 9802-9807; Zhao, M., Zen, K.-C., et al. (1999) Biochemistry 38, 7407-7412]. Since hydropathy plots indicate that these residues are at the membrane-water interface at the cytoplasmic surface of the membrane, site-directed nitroxide scanning electron paramag netic resonance (EPR) has been carried out on this region of the permease. Thirty-one single-Cys permease mutants were spin-labeled and examined by co nventional and power saturation EPR. The motional freedom of the side chain s, as well as accessibility to O-2 or potassium chromium oxalate (CrOx), in dicates that the loop between helices IV and V (loop IV/V) is considerably smaller than predicted by hydropathy plots, extending only from about Val13 2 to Phe138 and that Glu126 and Arg144 are probably within the membrane. Al though ligand binding has no effect on the mobility of the labeled side cha ins, a marked increase in CrOx and O-2 accessibility is observed at positio n 137, as well as significant changes in accessibility to CrOx on one face of helix V. It is concluded that ligand binding induces a conformational ch ange in the vicinity of the binding site, resulting in increased accessibil ity of position 137 in loop IV/V to solvent.