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 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) (