PMID:10625464
| Citation |
Zhao, M, Zen, KC, Hernandez-Borrell, J, Altenbach, C, Hubbell, WL and Kaback, HR (1999) Nitroxide scanning electron paramagnetic resonance of helices IV and V and the intervening loop in the lactose permease of Escherichia coli. Biochemistry 38:15970-7 |
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| Abstract |
Glu126 and Arg144 in helices IV and V, respectively, in the lactose permease of Escherichia coli, which play an indispensable role in substrate binding, are charge-paired and in close proximity [Venkatesan, P., Kaback, H. 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 paramagnetic resonance (EPR) has been carried out on this region of the permease. Thirty-one single-Cys permease mutants were spin-labeled and examined by conventional and power saturation EPR. The motional freedom of the side chains, as well as accessibility to O(2) or potassium chromium oxalate (CrOx), indicates that the loop between helices IV and V (loop IV/V) is considerably smaller than predicted by hydropathy plots, extending only from about Val132 to Phe138 and that Glu126 and Arg144 are probably within the membrane. Although ligand binding has no effect on the mobility of the labeled side chains, a marked increase in CrOx and O(2) accessibility is observed at position 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 change in the vicinity of the binding site, resulting in increased accessibility of position 137 in loop IV/V to solvent. |
| Links | |
| Keywords |
Amino Acid Sequence; Cyclic N-Oxides; Electron Spin Resonance Spectroscopy; Escherichia coli/enzymology; Escherichia coli Proteins; Ligands; Membrane Transport Proteins/chemistry; Mesylates; Models, Molecular; Molecular Sequence Data; Monosaccharide Transport Proteins; Protein Structure, Secondary; Protein Structure, Tertiary; Spin Labels; Symporters |
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