Outer membrane phospholipase A is dimeric in phospholipid bilayers: a cross-linking and fluorescence resonance energy transfer study.
In the cell, the activity of outer membrane phospholipase A (OMPLA) is strictly regulated to prevent uncontrolled breakdown of the membrane lipids. Previously, it has been shown that the enzymatic activity is modulated by reversible dimerization. The current studies were carried out to define the oligomeric state of OMPLA in a membrane and to investigate the activation process. Three single-cysteine variant proteins H26C, H234C, and S144C were produced and purified to homogeneity. Using maleimido-based homo-bifunctional cross-linking reagents, H26C could be efficiently cross-linked as assessed by SDS-PAGE, whereas S144C and H234C could not be cross-linked. These data suggest that residue 26 is located close to the dimer symmetry axis. H26C was specifically labeled with 5-({[(2-iodoacetyl)amino]ethyl}amino)naphthalene-1-sulfonic acid and N,N'-dimethyl-N-(iodoacetyl)-N-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl)ethylenediamine as the fluorescent energy donor and acceptor, respectively, and dimerization was investigated using fluorescence resonance energy transfer (FRET). Quenching of the donor in the presence of the acceptor demonstrated the dimeric nature of OMPLA, in agreement with cross-linking data. The observed FRET effect was dependent on the cofactor calcium, and the presence of substrate, indicating the specificity of the dimerization process. The labeled protein was reconstituted in phospholipid vesicles. In bilayers, OMPLA exhibited low activity and was dimeric as assessed by FRET. Addition of detergent resulted in a 70-fold increase in activity, while the protein remained dimeric. The results are discussed in terms of the activation of dimeric OMPLA due to changes in the physical state of the bilayer which occur upon perturbation of the membrane integrity.
Ubarretxena-Belandia
I
I
Department of Enzymology and Protein Engineering, Center for Biomembranes and Lipid Enzymology, Institute of Biomembranes, Utrecht University, The Netherlands.
Hozeman
L
L
van der Brink-van der Laan
E
E
Pap
E H
EH
Egmond
M R
MR
Verheij
H M
HM
Dekker
N
N
D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry* / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000737:chemistry*; D003902:Detergents; D019281:Dimerization; D004735:Energy Transfer; D005456:Fluorescent Dyes / Q000737:chemistry; D008051:Lipid Bilayers / Q000737:chemistry* / Q000378:metabolism; D008823:Micelles; D009282:Naphthalenesulfonates / Q000737:chemistry; D010713:Phosphatidylcholines / Q000737:chemistry; D010741:Phospholipases A / Q000737:chemistry* / Q000378:metabolism; D010743:Phospholipids / Q000737:chemistry* / Q000378:metabolism; D012486:Salmonella typhimurium / Q000201:enzymology; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D013439:Sulfhydryl Reagents / Q000737:chemistry
D001425:Bacterial Outer Membrane Proteins; D003432:Cross-Linking Reagents; D003902:Detergents; D005456:Fluorescent Dyes; D008051:Lipid Bilayers; D008823:Micelles; D009282:Naphthalenesulfonates; D010713:Phosphatidylcholines; D010743:Phospholipids; D013439:Sulfhydryl Reagents; C006421:1,5-I-AEDANS; D010741:Phospholipases A