Structural Information on a Membrane Transport Protein from Nuclear Magnetic Resonance Spectroscopy Using Sequence-Selective Nitroxide Labeling

• Published in: Biochemistry (Easton) 1999-07, Vol.38 (30), p.9634-9639
• Main Authors: Spooner, Paul J. R, Veenhoff, Liesbeth M, Watts, Anthony, Poolman, Bert
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Biological Transport - genetics; Carrier Proteins - chemistry; Carrier Proteins - genetics; Carrier Proteins - metabolism; Cell Membrane - enzymology; Cyclic N-Oxides - metabolism; Cysteine - genetics; Escherichia coli Proteins; Lysine - genetics; Maleimides - chemistry; Membrane Transport Proteins - chemistry; Membrane Transport Proteins - genetics; Membrane Transport Proteins - metabolism; Monosaccharide Transport Proteins; Nuclear Magnetic Resonance, Biomolecular - methods; Protein Binding - genetics; Spin Labels; Streptococcus - enzymology; Substrate Specificity - genetics; Symporters
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi990745l

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) (