title: Relative substrate affinities of wild-type and mutant forms of the Escherichia coli sugar transporter GalP determined by solid-state NMR.
abstract: Solid-state nuclear magnetic resonance (SSNMR) spectroscopy is used for the first time to examine the relative substrate-binding affinities of mutant forms of the Escherichia coli sugar transporter GalP in membrane preparations. The SSNMR method of (13)C cross-polarization magic-angle spinning (CP-MAS) is applied to five site-specific mutants (W56F, W239F, R316W, T336Y and W434F), which have a range of different sugar-transport activities compared to the wild-type protein. It is shown that binding of the substrate D-glucose can be detected independently of sugar transport activity using SSNMR, and that the NMR peak intensities for uniformly (13)C-labelled glucose are consistent with wild-type GalP and the mutants having different affinities for the substrate. The W239F and W434F mutants showed binding affinities similar to that of the wild-type protein, whereas the affinity of glucose-binding to the W56F mutant was reduced. The R316W mutant showed no detectable binding; this position corresponds to the second basic residue in the highly conserved (R/K)XGR(R/K) motif in the major facilitator superfamily of transport proteins and to a mutation in human GLUT1 found in individuals with GLUT1-deficiency syndrome. The T336Y mutant also showed no detectable binding; this mutation is likely to have perturbed helix structure or packing to an extent that conformational changes in the protein are hindered. The effects of the mutations on substrate-binding are discussed with reference to the putative positions of the residues in a 3D homology model of GalP based on the X-ray crystal structure of the E. coli glycerol-3-phosphate transporter GlpT.
authors.affiliation: Astbury Centre for Structural Molecular Biology and Institute of Membrane and Systems Biology, University of Leeds, Leeds, UK. s.g.patching@leeds.ac.uk
authors.forename: Simon G
authors.initials: SG
authors.lastname: Patching
authors.forename: Georgios
authors.initials: G
authors.lastname: Psakis
authors.forename: Stephen A
authors.initials: SA
authors.lastname: Baldwin
authors.forename: Jocelyn
authors.initials: J
authors.lastname: Baldwin
authors.forename: Peter J F
authors.initials: PJ
authors.lastname: Henderson
authors.forename: David A
authors.initials: DA
authors.lastname: Middleton
mesh_terms: D020816:Amino Acid Motifs; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002247:Carbon Isotopes; D017124:Conserved Sequence; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005947:Glucose / Q000378:metabolism; D051272:Glucose Transporter Type 1 / Q000172:deficiency / Q000235:genetics; D006801:Humans; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D009004:Monosaccharide Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020125:Mutation, Missense; D033902:Periplasmic Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D002135:Calcium-Binding Proteins; D002247:Carbon Isotopes; D029968:Escherichia coli Proteins; D051272:Glucose Transporter Type 1; D009004:Monosaccharide Transport Proteins; D033902:Periplasmic Binding Proteins; C495677:SLC2A1 protein, human; C023550:galactose-binding protein; D005947:Glucose