Mode of membrane interaction of wild-type and mutant signal peptides of the Escherichia coli outer membrane protein A.
The membrane insertion potentials of the signal peptide of the outer membrane protein A (OmpA) from Escherichia coli and two peptides corresponding to functionally impaired mutant OmpA signal sequences were examined using spin label electron spin resonance (ESR) spectroscopy. The wild-type OmpA signal peptide, WT, a deletion mutant lacking the amino acid stretch 6-9, delta 6-9, and a substitution mutant with the isoleucine residue at position 8 replaced by asparagine, I8N, were incorporated into mixed lipid vesicles containing negatively charged 1-palmitoyl-2-oleoyl phosphatidylglycerol (POPG) and zwitterionic 1-palmitoyl-2-oleoyl phosphatidylethanolamine (POPE). Spin-labeled derivatives of phosphatidylglycerol and phosphatidylethanolamine containing a nitroxide moiety at the 12th position in the sn-2 acyl chain, 12-PGSL and 12-PESL, respectively, were employed for the ESR experiments. The 12-PGSL and 12-PESL exhibited two-component spectra in the presence of the WT and delta 6-9, but not when I8N was present. Using difference spectroscopy, the number of POPG and POPE molecules associated with an ordered lipid layer surrounding the peptides was estimated. The results suggest that WT exists as a transmembrane monomer in the membrane. The delta 6-9 mutant signal peptide appears to exist either as a transmembrane aggregate or partially inserted into the acyl chain region. The substitution mutant, I8N, has a most probable location near the membrane surface. Among these variants of the OmpA signal peptide, the ability to adopt a transmembrane monomeric orientation correlates well with the export activity.
Sankaram
M B
MB
Department of Biochemistry, University of Virginia Health Sciences Center, Charlottesville 22908-0001.
Jones
J D
JD
D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli; D008969:Molecular Sequence Data; D010714:Phosphatidylethanolamines / Q000737:chemistry; D010715:Phosphatidylglycerols / Q000737:chemistry; D021382:Protein Sorting Signals / Q000737:chemistry*; D013329:Structure-Activity Relationship
D001425:Bacterial Outer Membrane Proteins; D010714:Phosphatidylethanolamines; D010715:Phosphatidylglycerols; D021382:Protein Sorting Signals