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The lactose transport protein (LacS) from Streptococcus thermophilus bearing a single cysteine mutation, K373C, within the putative interhelix loop 10-11 has been overexpressed in native membranes. Cross-polarization magic-angle spinning nuclear magnetic resonance spectroscopy (NMR) could selectively distinguish binding of (13)C-labeled substrate to just 50-60 nmol of LacS(K373C) in the native fluid membranes. Nitroxide electron spin-label at the K373C location was essentially immobile on the time scale of both conventional electron spin resonance spectroscopy (ESR) (<10(-8)s) and saturation-transfer ESR (<10(-3)s), under the same conditions as used in the NMR studies. The presence of the nitroxide spin-label effectively obscured the high-resolution NMR signal from bound substrate, even though (13)C-labeled substrate was shown to be within the binding center of the protein. The interhelix loop 10-11 is concluded to be in reasonably close proximity to the substrate binding site(s) of LacS (<15 A), and the loop region is expected to penetrate between the transmembrane segments of the protein that are involved in the translocation process.

Original publication

DOI

10.1021/bi990745l

Type

Journal article

Journal

Biochemistry

Publication Date

27/07/1999

Volume

38

Pages

9634 - 9639

Keywords

Amino Acid Sequence, Biological Transport, Carrier Proteins, Cell Membrane, Cyclic N-Oxides, Cysteine, Escherichia coli Proteins, Lysine, Maleimides, Membrane Transport Proteins, Monosaccharide Transport Proteins, Nuclear Magnetic Resonance, Biomolecular, Protein Binding, Spin Labels, Streptococcus, Substrate Specificity, Symporters