Structural information on a membrane transport protein from nuclear magnetic resonance spectroscopy using sequence-selective nitroxide labeling, Biochemistry 38

ABSTRACT: 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 13C-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) (<10-8s) and saturation-transfer ESR (<10-3s), 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 13C-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 Å), and the loop region is expected to penetrate between the transmembrane segments of the protein that are involved in the translocation process. The lactose transport protein (LacS) of Streptococcus thermophilus belongs to the class of secondary transport systems comprising several dozen families to date (1, 2)