Specific spin labelling of the sugar-H+ symporter, GalP, in cell membranesof Escherichia coli: site mobility and overall rotational diffusion of theprotein
D. Marsh et Pjf. Henderson, Specific spin labelling of the sugar-H+ symporter, GalP, in cell membranesof Escherichia coli: site mobility and overall rotational diffusion of theprotein, BBA-BIOMEMB, 1510(1-2), 2001, pp. 464-473
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.