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)
Rodionova
N A
NA
Department of Molecular Physiology and Biological Physics, University of Virginia School of Medicine, Charlottesville 22908.
Tatulian
S A
SA
Surrey
T
T
Jähnig
F
F
Tamm
L K
LK
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