Specific spin labelling of the sugar-H+ symporter, GalP, in cell membranesof Escherichia coli: site mobility and overall rotational diffusion of theprotein

The D-galactose-H+ symport protein (GalP) of Escherichia coli is a homologu e of the human glucose transport protein, GLUTI. After amplified expression of the GalP transporter in E. coli, other membrane proteins were prereacte d with N-ethylmaleimide in the presence of excess D-galactose to protect Ga lP. Inner membranes were then specifically spin labelled on Cys(374) Of Gal P with 4-maleimide-2,2,6,6-tetramethylpiperidin-1-oxyl. The electron parama gnetic resonance (EPR) spectra are characteristic of a single labelling sit e in which the mobility of the spin label is very highly constrained. This is confirmed with other nitroxyl spin labels, which are derivatives of iodo acetamide and indanedione. Saturation transfer EPR spectra indicate that th e overall rotation of the GalP protein in the membrane is slow at low tempe ratures (approx. 2 degreesC), but considerably more rapid and highly anisot ropic at physiological temperatures. The rate of rotation about the membran e normal at 37 degreesC is consistent with predictions for a 12-transmembra ne helix assembly that is less than closely packed. (C) 2001 Elsevier Scien ce B.V. All rights reserved.