9634
Biochemistry 1999, 38, 9634-9639
Structural Information on a Membrane Transport Protein from Nuclear Magnetic Resonance Spectroscopy Using Sequence-Selective Nitroxide Labeling† Paul J. R. Spooner,*,‡ Liesbeth M. Veenhoff,§ Anthony Watts,‡ and Bert Poolman§ Biomembrane Structure Unit, Department of Biochemistry, UniVersity of Oxford, South Parks Road, Oxford OX1 3QU, United Kingdom, and Department of Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, UniVerstity of Groningen, Kerklaan 30, 9751 NN Haren, The Netherlands ReceiVed March 31, 1999
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) (
