Characterization of two membrane-bound forms of OmpA.
The insertion of the outer membrane protein A (OmpA) into lipid bilayers was studied by limited proteolysis, polarized Fourier transform infrared (FTIR) spectroscopy, and fluorescence spectroscopy. In the native state, OmpA is thought to form a barrel of eight antiparallel beta-strands. For the present study, it was isolated in an unfolded form, purified, and exposed to performed vesicles of 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC), dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), and three phospholipids that were brominated in different positions of their sn-2 chains (4,5-BrPC, 9,10-BrPC, and 11,12-BrPC). Limited proteolysis revealed two membrane-bound forms of OmpA, namely an "adsorbed" (35 kDa) and an "inserted" (30 kDa) form [Surrey, T., & Jähnig, F. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 7457-7461]. Which form was found after membrane binding and refolding depended on the lipids used and on the temperature. Polarized attenuated total reflection (ATR)-FTIR spectra were recorded with OmpA bound to germanium-supported bilayers in both forms. The position of the amide I' band indicated quite large fractions of beta-structure of OmpA in both membrane-bound forms (35-45% in the adsorbed form and 45-55% in the inserted form). Measurements of the linear dichroism of the amide I' bands in the inserted form are consistent with an antiparallel beta-barrel in which the strands are inclined at about 36 degrees from the membrane normal. The average angle of the beta-strands to the bilayer normal is likely larger in the 35 kDa form than in the inserted form.(ABSTRACT TRUNCATED AT 250 WORDS)
Department of Molecular Physiology and Biological Physics, University of Virginia School of Medicine, Charlottesville 22908.
N A
NA
Rodionova
S A
SA
Tatulian
T
T
Surrey
F
F
Jähnig
L K
LK
Tamm
D015060:1,2-Dipalmitoylphosphatidylcholine / Q000378:metabolism; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D001966:Bromine; D004134:Dimyristoylphosphatidylcholine / Q000378:metabolism; D004926:Escherichia coli / Q000737:chemistry; D008051:Lipid Bilayers / Q000378:metabolism*; D008081:Liposomes / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism; D010713:Phosphatidylcholines / Q000737:chemistry / Q000378:metabolism; D017510:Protein Folding; D017433:Protein Structure, Secondary; D013050:Spectrometry, Fluorescence; D017550:Spectroscopy, Fourier Transform Infrared; D014357:Trypsin / Q000378:metabolism
D001425:Bacterial Outer Membrane Proteins; D008051:Lipid Bilayers; D008081:Liposomes; D010446:Peptide Fragments; D010713:Phosphatidylcholines; D015060:1,2-Dipalmitoylphosphatidylcholine; D014357:Trypsin; D001966:Bromine; C028694:1-palmitoyl-2-oleoylphosphatidylcholine; D004134:Dimyristoylphosphatidylcholine