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 bearin g a single cysteine mutation, K373C, within the putative interhelix loop 10 -11 has been overexpressed in native membranes. Cross-polarization magic-an gle spinning nuclear magnetic resonance spectroscopy (NMR) could selectivel y distinguish binding of C-13-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 conventio nal electron spin resonance spectroscopy (ESR) (<10(-8)s) and saturation-tr ansfer 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-re solution NMR signal from bound substrate, even though C-13-labeled substrat e 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 substrat e binding site(s) of LacS (<15 Angstrom), and the loop region is expected t o penetrate between the transmembrane segments of the protein that are invo lved in the translocation process.