Showing papers by "B. de Kruijff published in 1993"

Characterization of a Bacillus subtilis SecA mutant protein deficient in translocation ATPase and release from the membrane

Abstract: SecA is the precursor protein binding subunit of the bacterial precursor protein translocase, which consists of the SecY/E protein as integral membrane domain. SecA is an ATPase, and couples the hydrolysis of ATP to the release of bound precursor proteins to allow their proton-motive-force-driven translocation across the cytoplasmic membrane. A putative ATP-binding motif can be predicted from the amino acid sequence of SecA with homology to the consensus Walker A-type motif. The role of this domain is not known. A lysine residue at position 106 at the end of the glycine-rich loop in the A motif of the Bacillus subtilis SecA was replaced by an asparagine through site-directed mutagenesis (K106N SecA). A similar replacement was introduced at an adjacent lysine residue at position 101 (K101N SecA). Wild-type and mutant SecA proteins were expressed to a high level and purified to homogeneity. The catalytic efficacy (kcat/km) of the K106N SecA for lipid-stimulated ATP hydrolysis was only 1% of that of the wild-type and K101N SecA. K106N SecA retained the ability to bind ATP, but its ATPase activity was not stimulated by precursor proteins. Mutant and wild-type SecA bind with similar affinity to Escherichia coli inner membrane vesicles and insert into a phospholipid monolayer. In contrast to the wild type, membrane insertion of the K106N SecA was not prevented by ATP. K106N SecA blocks the ATP and proton-motive-force-dependent chase of a translocation intermediate to fully translocated proOmpA. It is concluded that the GKT motif in the amino-terminal domain of SecA is part of the catalytic ATP-binding site. This site may be involved in the ATP-driven protein recycling function of SecA which allows the release of SecA from its association with precursor proteins, and the phospholipid bilayer.

Chapter 5 Lipid-peptide interactions in model systems: Membrane insertion and translocation of peptides

Abstract: Publisher Summary Lipid–peptide/protein interactions determine the structure and function of biomembranes. These interactions play an important role in both the activity of membrane-bound enzymes and the transport of solutes across membranes. There is growing experimental evidence that the interaction of peptides or proteins with the lipid phase of biological membranes plays a role in processes such as membrane insertion and the translocation of precursor proteins. The study of peptide–lipid model systems provides information on the possibilities and limitations of the putative function of peptide–lipid interactions in various biological processes. This chapter presents an overview of the results that have emerged from the integrated approach that is chosen to study the molecular details of membrane insertion and the translocation of peptides. The main parameters affecting peptide–lipid interactions have been investigated in well-defined systems consisting of synthetic model peptides and phospholipid vesicles. This enabled a coherent analysis of the influence of the peptide's charge and hydrophobicity, of the membrane surface charge, and of the presence of transmembrane ion gradients that give rise to pH gradients and membrane potentials on lipid–peptide interaction.