Structural information on a membrane transport protein from nuclear magnetic resonance spectroscopy using sequence-selective nitroxide labeling.
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.
Spooner
P J
PJ
Biomembrane Structure Unit, Department of Biochemistry, University of Oxford, United Kingdom.
Veenhoff
L M
LM
Watts
A
A
Poolman
B
B
D000595:Amino Acid Sequence; D001692:Biological Transport / Q000235:genetics; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002462:Cell Membrane / Q000201:enzymology; D003497:Cyclic N-Oxides / Q000378:metabolism*; D003545:Cysteine / Q000235:genetics; D029968:Escherichia coli Proteins; D008239:Lysine / Q000235:genetics; D008301:Maleimides / Q000737:chemistry; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D009004:Monosaccharide Transport Proteins; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D011485:Protein Binding / Q000235:genetics; D013113:Spin Labels; D013291:Streptococcus / Q000201:enzymology; D013379:Substrate Specificity / Q000235:genetics; D027981:Symporters
D002352:Carrier Proteins; D003497:Cyclic N-Oxides; D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D008301:Maleimides; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D013113:Spin Labels; D027981:Symporters; C021499:lactose permease; D008239:Lysine; D003545:Cysteine