Fluorescence analysis of tryptophan-containing variants of the LamB signal sequence upon insertion into a lipid bilayer.
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.
McKnight
C J
CJ
Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas 75235.
Rafalski
M
M
Gierasch
L M
LM
D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins; D004926:Escherichia coli / Q000235:genetics; D005456:Fluorescent Dyes; D014644:Genetic Variation; D008051:Lipid Bilayers / Q000737:chemistry*; D008969:Molecular Sequence Data; D018272:Porins; D011487:Protein Conformation; D015202:Protein Engineering; D021382:Protein Sorting Signals / Q000138:chemical synthesis / Q000737:chemistry* / Q000235:genetics; D011991:Receptors, Virus / Q000138:chemical synthesis / Q000737:chemistry* / Q000235:genetics; D013050:Spectrometry, Fluorescence; D014364:Tryptophan
D001425:Bacterial Outer Membrane Proteins; D005456:Fluorescent Dyes; D008051:Lipid Bilayers; D018272:Porins; D021382:Protein Sorting Signals; D011991:Receptors, Virus; C016532:maltoporins; D014364:Tryptophan