Rotational mobility and orientational stability of a transport protein in lipid membranes.
A single-cysteine mutant of the lactose transport protein LacS(C320A/W399C) from Streptococcus thermophilus was selectively labeled with a nitroxide spin label, and its mobility in lipid membranes was studied as a function of its concentration in the membrane by saturation-transfer electron spin resonance. Bovine rhodopsin was also selectively spin-labeled and studied to aid the interpretation of the measurements. Observations of spin-labeled proteins in macroscopically aligned bilayers indicated that the spin label tends to orient so as to reflect the transmembrane orientation of the protein. Rotational correlation times of 1-2 micros for purified spin-labeled bovine rhodopsin in lipid membranes led to viscosities of 2.2 poise for bilayers of dimyristoylphosphatidylcholine (28 degrees C) and 3.0 poise for the specific mixture of lipids used to reconstitute LacS (30 degrees C). The rotational correlation time for LacS did not vary significantly over the range of low concentrations in lipid bilayers, where optimal activity was seen to decrease sharply and was determined to be 9 +/- 1 micros (mean +/- SD) for these samples. This mobility was interpreted as being too low for a monomer but could correspond to a dimer if the protein self-associates into an elongated configuration within the membrane. Rather than changing its oligomeric state, LacS appeared to become less ordered at the concentrations in aligned membranes exceeding 1:100 (w/w) with respect to the lipid.
Spooner
P J
PJ
Biomembrane Structure Unit, Department of Biochemistry, University of Oxford, Oxford OX1 3QU, United Kingdom. spooner@biochem.ox.ac.uk
Friesen
R H
RH
Knol
J
J
Poolman
B
B
Watts
A
A
D019943:Amino Acid Substitution; D000818:Animals; D002417:Cattle; D002462:Cell Membrane / Q000201:enzymology; D004134:Dimyristoylphosphatidylcholine; D004578:Electron Spin Resonance Spectroscopy; D029968:Escherichia coli Proteins; D008081:Liposomes; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D009004:Monosaccharide Transport Proteins; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012243:Rhodopsin / Q000737:chemistry* / Q000302:isolation & purification; D012399:Rotation; D013113:Spin Labels; D013291:Streptococcus / Q000201:enzymology; D027981:Symporters
D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D008081:Liposomes; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D011994:Recombinant Proteins; D013113:Spin Labels; D027981:Symporters; D012243:Rhodopsin; C021499:lactose permease; D004134:Dimyristoylphosphatidylcholine