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We have synthesized a peptide corresponding to the 25-residue signal sequence plus the first 28 residues of the Escherichia coli outer membrane protein LamB in order to explore the properties of a signal sequence in the presence of the N-terminal region of its passenger. In the last few years, there have been several observations of differing efficiencies of export when signal sequences are attached to different passenger proteins or when the first part of a passenger protein undergoes mutation. In the LamB case, gene fusions with lacZ have shown that the signal sequence plus the first 28 residues of mature LamB are necessary to direct beta-galactosidase into the export pathway [Rasmussen, B. A. & Silhavy, T. J. (1987) Genes Dev. 1, 185-196]. The origin of these observations and whether there is an influence of the mature region on the properties of the signal sequence have not been known. We find that the conformational and membrane-binding properties of the LamB signal sequence manifest in a 25-residue peptide are essentially unaltered in the context of the 53-residue peptide corresponding to this signal sequence plus the first 28 residues of the mature LamB protein. CD spectra show that the signal peptide and passenger domains are conformationally independent of each other in micelle or bilayer environments. Furthermore, the signal sequence leads to the spontaneous association of the 53-residue peptide with a lipid bilayer; alone, the mature domain does not interact with lipid bilayers. Fluorescence results show that the mode of interaction of the signal peptide with a bilayer is essentially unaltered by the presence of its mature region. This lack of influence of the mature domain on the behavior of the signal sequence is unexpected for juxtaposed polypeptides of comparable length and may be of physiological importance: N-terminal regions of secreted proteins may be selected to be passive, by comparison with their cognate signal sequences, which themselves must engage the export apparatus and actively interact with its components.

To investigate the interaction of the LamB signal sequence with lipid bilayers, we have synthesized three tryptophan-containing analogues of the wild-type signal peptide. The tryptophan residues were used as intrinsic fluorescent probes of the N-terminal (position 5), central (position 18), and C-terminal (position 24) regions of the 25-residue peptide. The tryptophan substitutions did not significantly alter the physical properties of the wild-type signal peptide. In the presence of lipid vesicles which mimic the composition of the Escherichia coli inner membrane, the peptides adopt alpha-helical structure, and the tryptophan fluorescence emission maximum is shifted to shorter wavelength, indicating that the peptides insert into the acyl chain region of the lipid bilayer. Fluorescence quenching by soluble, aqueous-phase (I-), and membrane-resident (nitroxide-labeled lipids) quenchers was used to locate the tryptophans in each peptide within the bilayer. The C-terminus was interfacial while the central region of the signal sequence was deeply buried within the acyl chain region of the bilayer. The tryptophan at position 5 was buried but less deeply than the tryptophan at position 18. This topology is consistent with either a looped or a transmembrane orientation of signal peptide. However, either structure must accommodate the high helical content of the peptides in vesicles. These results indicate that the LamB signal sequence spontaneously inserts into the acyl chain region of lipid membranes in the absence of any of the proteins involved in protein secretion.

A detailed nuclear magnetic resonance analysis of the isolated LamB signal peptide (MMITLRKLPLAVAVAAGVMSAQAMA) under conditions defined by circular dichroism spectra to mimic the conformational distribution of this peptide in membranelike environments has provided a description of specific residue conformational preferences. This 25-residue long peptide in 20 mol % trifluoroethanol in water is in dynamic equilibrium between a helical and a more random conformation, and this equilibrium is shifted toward the more random structure as the temperature is raised. Part of the molecule, residues 10-18, exists in a stable helix at all temperatures studied (5, 25, and 50 degrees C). Propagation of the helix through the C-terminal end occurs at 25 degrees C, while the temperature must be lowered to 5 degrees C to observe any significant population of a helical conformation in the N-terminal region. These results argue that the Pro and Gly residues, which flank the helical segment, act to disfavor helix propagation on their N- or C-terminal sides, respectively. The influence of the Pro residue is stronger than that of the Gly. Furthermore, the most stable part of the helix in this signal peptide under the conditions studied is the hydrophobic core, which is the hallmark of functional signal sequences.

The interaction of a chemically synthesized 25-residue signal peptide of LamB protein from Escherichia coli with phospholipids has been studied with a film balance technique. The conformation, orientation, and concentration of the peptides in lipid monolayers have been determined from polarized infrared spectroscopy, ultraviolet spectroscopy, and assay of 14C-labeled peptide in transferred films. When the LamB signal peptide is injected into the subphase under a phosphatidylethanolamine-phosphatidylglycerol monolayer at low initial pressure, insertion of a portion of the peptide into the lipid film is evidenced by a rapid rise in film pressure. Spectroscopic results obtained on films transferred to quartz plates and Ge crystals show that the peptide is a mixture of alpha-helix and beta-conformation where the long axis of the alpha-helix penetrates the monolayer plane and the beta-structure is coplanar with the film. By contrast, when peptide is injected under lipid at high initial pressure, no pressure rise is observed, and the spectroscopic results show the presence of only beta-structure which is coplanar with the monolayer. The spectroscopic and radioassay results are all consistent with the picture of a peptide anchored to the monolayer through electrostatic binding with a helical portion inserted into the lipid region of the monolayer and a beta-structure portion resident in the aqueous phase. The negative charges on the lipid molecules are roughly neutralized by the positive charges of the peptide.

The role of the signal sequence in the secretion of proteins remain unclear despite extensive research. We have examined properties of synthetic peptides corresponding to a family of signal sequences derived from the lamB gene of Escherichia coli, including five examples of known phenotype that contain mutations in the signal sequence. By circular dichroism spectroscopy, the wild type and export-component mutant signal peptides show a high alpha-helix content in membrane mimetic environments (sodium dodecyl sulfate micelles and phospholipid vesicles). Tendency to adopt helical conformations is clearly not sufficient to define a functional signal sequence, however, as some nonfunctional mutant signal peptides also contain a relatively high proportion of alpha-helix. The affinity of these peptides for phospholipid monolayers as assessed by surface tensiometry reveals further distinguishing properties. Export-competent peptides show an increased affinity for and greater perturbation of phospholipid monolayers and bilayers than to export-defective peptides. These results suggest a lipid binding role for the signal sequence during protein export in addition to its recognition by proteins of the export pathway.