Specific spin labelling of the sugar-H(+) symporter, GalP, in cell membranes of Escherichia coli: site mobility and overall rotational diffusion of the protein.
The D-galactose-H(+) symport protein (GalP) of Escherichia coli is a homologue of the human glucose transport protein, GLUT1. After amplified expression of the GalP transporter in E. coli, other membrane proteins were prereacted with N-ethylmaleimide in the presence of excess D-galactose to protect GalP. Inner membranes were then specifically spin labelled on Cys(374) of GalP with 4-maleimide-2,2,6,6-tetramethylpiperidine-1-oxyl. The electron paramagnetic resonance (EPR) spectra are characteristic of a single labelling site in which the mobility of the spin label is very highly constrained. This is confirmed with other nitroxyl spin labels, which are derivatives of iodoacetamide and indanedione. Saturation transfer EPR spectra indicate that the overall rotation of the GalP protein in the membrane is slow at low temperatures (approx. 2 degrees C), but considerably more rapid and highly anisotropic at physiological temperatures. The rate of rotation about the membrane normal at 37 degrees C is consistent with predictions for a 12-transmembrane helix assembly that is less than closely packed.
Marsh
D
D
Max-Planck-Institut für biophysikalische Chemie, Göttingen, Germany. dmarsh@gwdg.de
Henderson
P J
PJ
D000595:Amino Acid Sequence; D001665:Binding Sites; D002135:Calcium-Binding Proteins; D002462:Cell Membrane / Q000378:metabolism; D004058:Diffusion; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D033902:Periplasmic Binding Proteins; D010957:Plasmids; D013113:Spin Labels; D013696:Temperature
D002135:Calcium-Binding Proteins; D009004:Monosaccharide Transport Proteins; D033902:Periplasmic Binding Proteins; D013113:Spin Labels; C023550:galactose-binding protein