title: Selective NMR observation of inhibitor and sugar binding to the galactose-H(+) symport protein GalP, of Escherichia coli.
abstract: The binding of the transport inhibitor forskolin, synthetically labelled with (13)C, to the galactose-H(+) symport protein GalP, overexpressed in its native inner membranes from Escherichia coli, was studied using cross-polarization magic angle spinning (13)C NMR. (13)C-Labelled D-galactose and D-glucose were displaced from GalP with the singly labelled [7-OCO(13)CH(3)]forskolin and were not bound to any alternative site within the protein, demonstrating that any multiple sugar binding sites are not simultaneously accessible to these sugars and the inhibitor within GalP. The observation of singly (13)C-labelled forskolin was hampered by interference from natural abundance (13)C in the membranes and so the effectiveness of double-quantum filtration was assessed for the exclusive detection of (13)C spin pairs in sugar (D-[1,2-(13)C(2)]glucose) and inhibitor ([7-O(13)CO(13)CH(3)]forskolin) bound to the GalP protein. The solid state NMR methodology was not effective in creating double-quantum selection of ligand bound with membranes in the 'fluid' state (approx. 2 degrees C) but could be applied in a straightforward way to systems that were kept frozen. At -35 degrees C, double-quantum filtration detected unbound sugar that was incorporated into ice structure within the sample, and was not distinguished from protein-bound sugar. However, the method detected doubly labelled forskolin that is selectively bound only to the transport system under these conditions and provided very effective suppression of interference from natural abundance (13)C background. These results indicate that solid state NMR methods can be used to resolve selectively the interactions of more hydrophobic ligands in the binding sites of target proteins.
authors: Biomembrane Structure Unit, Department of Biochemistry, University of Oxford, UK.
authors: A N
authors: AN
authors: Appleyard
authors: R B
authors: RB
authors: Herbert
authors: P J
authors: PJ
authors: Henderson
authors: A
authors: A
authors: Watts
authors: P J
authors: PJ
authors: Spooner
mesh_terms: D002135:Calcium-Binding Proteins; D002247:Carbon Isotopes; D005576:Colforsin / Q000494:pharmacology; D004926:Escherichia coli / Q000737:chemistry / Q000378:metabolism*; D005615:Freezing; D005690:Galactose / Q000378:metabolism; D005947:Glucose / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008565:Membrane Proteins / Q000378:metabolism*; D015394:Molecular Structure; D009004:Monosaccharide Transport Proteins / Q000037:antagonists & inhibitors* / Q000096:biosynthesis / Q000378:metabolism; D033902:Periplasmic Binding Proteins
chemical_list: D002135:Calcium-Binding Proteins; D002247:Carbon Isotopes; D008565:Membrane Proteins; D009004:Monosaccharide Transport Proteins; D033902:Periplasmic Binding Proteins; C023550:galactose-binding protein; D005576:Colforsin; D005947:Glucose; D005690:Galactose