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 m embrane protein A (OmpA) from Escherichia coli and two peptides corres ponding to functionally impaired mutant OmpA signal sequences were exa mined using spin label electron spin resonance (ESR) spectroscopy. The wild-type OmpA signal peptide, WT, a deletion mutant lacking the amin o acid stretch 6-9, Delta 6-9, and a substitution mutant with the isol eucine residue at position 8 replaced by asparagine, I8N, were incorpo rated into mixed lipid vesicles containing negatively charged 1-palmit oyl-2-oleoyl phosphatidylglycerol (POPG) and zwitterionic 1-palmitoyl- 2-oleoyl phosphatidylethanolamine (POPE). Spin-labeled derivatives of phosphatidylglycerol and phosphatidylethanolamine containing a nitroxi de 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 spectro scopy, the number of POPG and POPE molecules associated with an ordere d lipid layer surrounding the peptides was estimated. The results sugg est that WT exists as a transmembrane monomer in the membrane. The Del ta 6-9 mutant signal peptide appears to exist either as a transmembran e aggregate or partially inserted into the acyl chain region. The subs titution mutant, I8N, has a most probable location near the membrane s urface. Among these variants of the OmpA signal peptide, the ability t o adopt a transmembrane monomeric orientation correlates well with the export activity.