Abstract
A signal peptide is required for the entry of a protein into the secretory pathway but how it functions in concert with the other transport components to achieve protein localization is not known. In Escherichia coli, SecA is a component of the transport machinery which may play a role in targeting the preprotein to membrane-bound translocation sites and then utilize the energy of ATP hydrolysis to initiate membrane insertion of the preprotein. This model requires that the signal peptide interact specifically with SecA and that features of the signal peptide promote binding. These issues were examined using a wild type synthetic signal sequence derived from E. coli alkaline phosphatase and several model signal peptides that differ in amino-terminal charge, core region hydrophobicity, and the ability to form an α-helical structure. Using a SecA/lipid ATPase assay as an indicator of binding, we observe maximum activity with the functional wild type peptide, 3K7L and 1K7L; these have very hydrophobic core regions and a high propensity for α-helix formation, while no significant reactions were noted for the non-functional peptides, 3K2L and 1K2L. Although peptides of intermediate hydrophobicity, 3K4L and 1K4L, both stimulated the SecA ATPase activity to an intermediate extent, the level of stimulation was more marked with the 3K4L peptide. This is consistent with in vivo analyses which indicate that for signal peptides of intermediate hydrophobicity, the amino terminal basic residues also play a key role in enhancing transport activity. The data suggest that signal peptide core region hydrophobicity, amino-terminal charge, and α-helicity contribute to the modulation of SecA/lipid ATPase activity by altering the binding affinity of the peptide for SecA. Separately, a competition binding assay was employed to establish that signal peptides also interact with SecA in aqueous solution. Furthermore, the interaction of functional signal peptides with SecA alters the SecA conformation sufficiently, for both soluble and membrane-associated forms, to cause a marked change in its V8 protease sensitivity.
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Miller, A., Wang, L., Kendall, D.A. (2002). Model Signal Peptides: Probes of Molecular Interactions During Protein Secretion. In: Self-Assembling Peptide Systems in Biology, Medicine and Engineering. Springer, Dordrecht. https://doi.org/10.1007/0-306-46890-5_15
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DOI: https://doi.org/10.1007/0-306-46890-5_15
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