Showing papers by "B. de Kruijff published in 1996"

Mode of Insertion of the Signal Sequence of a Bacterial Precursor Protein into Phospholipid Bilayers As Revealed by Cysteine-Based Site-Directed Spectroscopy

Abstract: The interactions between a bacterial precursor protein and phospholipids in bilayer-based model membrane systems is addressed in this study. The precursor-lipid interactions were assessed from the side of the lipid phase by fluorescence and electron spin resonance spectroscopy, using the precursor of the Escherichia coli outer membrane protein PhoE. The role of the signal sequence, as part of the precursor, in this interaction was investigated by using cysteine-based site-directed spectroscopy. For this purpose, purified cysteine-containing mutants of prePhoE, which were made by site-directed mutagenesis of the signal sequence part and of the mature part, and defined lipids were used. The location of the fluorescently labeled cysteine residues was established by resonance energy transfer and quenching experiments and those of the corresponding spin-labeled cysteine residues by paramagnetic relaxation enhancement. It was demonstrated that precursor-phospholipid interactions exist in model membrane systems and also that these interactions were dependent on the presence of anionic phospholipids and resulted in a deep insertion of (parts of) the precursor into the lipid bilayer. Furthermore, the results with the cysteine mutations in the signal sequence of the precursor indicate that both termini of the signal sequence are located near or at the membrane surface, with only the fluorescence of the labeled cysteines in the signal sequence part being protected against aqueous quenchers. The results demonstrate that, when part of the intact precursor, the signal sequence experiences similar lipid-protein interactions as do isolated signal peptides. They also indicate that the signal sequence inserts entirely as a looped structure into the membrane. In addition, the data also indicate that the mature part of the precursor has an affinity for the membrane.

Delta mu H+ dependency of in vitro protein translocation into Escherichia coli inner-membrane vesicles varies with the signal-sequence core-region composition.

Abstract: Signal sequences frequently contain alpha-helix-destabilizing amino acids in the hydrophobic core. Nuclear magnetic resonance studies on the conformation of signal sequences in membrane mimetic environments revealed that these residues cause a break in the alpha-helix. In the precursor of the Escherichia coli outer membrane protein PhoE (pre-PhoE), a glycine residue at position -10 (Gly -10) is thought to be responsible for the break in the alpha-helix. We investigated the role of this glycine residue in the translocation process by employing site-directed mutagenesis. SDS-PAGE analysis showed drastic variations in the electrophoretic mobilities of the mutant precursor proteins, suggesting an important role of the glycine residue in determining the conformation of the signal sequence. In vivo, no drastic differences in the translocation kinetics were observed as compared with wild-type PhoE, except when a charged residue (Arg) was substituted for Gly -10. However, the in vitro translocation of all mutant proteins into inverted inner-membrane vesicles was affected. Two classes of precursors could be distinguished. Translocation of one class of mutant proteins (Ala, Cys and Leu for Gly -10) was almost independent of the presence of a delta mu H+, whereas translocation of the other class of precursors (wild type or Ser) was strongly decreased in the absence of the delta mu H+. Apparently, the delta mu H+ dependency of in vitro protein translocation varies with the signal-sequence core-region composition. Furthermore, a proline residue at position -10 resulted in a signal sequence that did not prevent the folding of the precursor in an in vitro trimerization assay.