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

Citation
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
Citations number
27
Categorie Soggetti
Biochemistry & Biophysics
Journal title
BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES
ISSN journal
00052736 → ACNP
Volume
1510
Issue
1-2
Year of publication
2001
Pages
464 - 473
Database
ISI
SICI code
0005-2736(20010209)1510:1-2<464:SSLOTS>2.0.ZU;2-#
Abstract
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.