Secondary structure and oligomerization of the E. coli glycerol facilitator.
The Major Intrinsic Proteins are found throughout the bacterial, plant, and animal kingdoms and are responsible for the rapid transport of water and other small, polar solutes across membranes. The superfamily includes the aquaporins, the aquaglyceroporins, and the glycerol facilitators. We have overexpressed and purified the Escherichia coli inner membrane glycerol facilitator. Approximately 7.5 mg of 95% pure protein is obtained from 1 L of Escherichia coli cells using immobilized metal affinity chromatography. Well-resolved matrix-assisted laser desorption ionization mass spectra were obtained by solubilization of the protein in octyl-beta-D-glucopyranoside (M(r) = 33 650.3; error approximately 0.4%). The recombinant glycerol facilitator is inserted into the bacterial inner membrane, is functional, and is inhibited by HgCl(2). Polyacrylamide gel electrophoresis suggests that the facilitator is predominantly monomeric when solubilized with dodecyl-beta-D-maltoside, octyl-beta-D-glucopyranoside, and sodium dodecyl sulfate, but that it self-associates, forming soluble oligomers when urea is used during extraction. Similar oligomeric species are demonstrated to exist in the bacterial membrane by chemical cross-linking experiments. Circular dichroism analysis shows that the protein is predominantly alpha-helical. Helix content is significantly higher in protein prepared in the absence of urea (42-55%) than in its presence (32%). A possible role for the facilitator oligomers in interactions with, and regulation of, the glycerol kinase is discussed.
Manley
D M
DM
Department of Chemistry, University of Manitoba, Winnipeg, Manitoba R3T 2N2, Canada.
McComb
M E
ME
Perreault
H
H
Donald
L J
LJ
Duckworth
H W
HW
O'Neil
J D
JD
D020346:Aquaporins; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001692:Biological Transport; D002462:Cell Membrane / Q000378:metabolism; D002942:Circular Dichroism; D003432:Cross-Linking Reagents / Q000737:chemistry; D003902:Detergents; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins; D005990:Glycerol / Q000378:metabolism*; D008970:Molecular Weight; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012995:Solubility; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013388:Succinimides / Q000737:chemistry
D020346:Aquaporins; D001425:Bacterial Outer Membrane Proteins; D003432:Cross-Linking Reagents; D003902:Detergents; D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; D013388:Succinimides; C096464:disuccinimidyl glutarate; C078043:GlpF protein, E coli; D005990:Glycerol; C019358:disuccinimidyl suberate