Enhancement of protein translocation across the membrane by specific mutations in the hydrophobic region of the signal peptide.

The hydrophobic region of the signal peptide of the OmpA protein of the Escherichia coli outer membrane was extensively altered in its hydrophobicity and predicted secondary structure by site-specific mutagenesis. The mutated signal peptides were fused to nuclease A from Staphylococcus aureus, and the function of the signal peptide was examined by measuring the rate of processing of the signal peptide. Six of the 12 mutated signal peptides in the nuclease hybrid were processed faster than the wild-type. In particular, the processing of the mutated signal peptide in which the alanine residue at position 9 was substituted with a valine residue was enhanced almost twofold over the processing of the wild-type signal peptide. In addition, the production of nuclease A fused with this mutated signal peptide also increased twofold. However, these effects were not observed when the mutated signal peptide was fused to TEM beta-lactamase. Analysis of the present mutations suggests that both overall hydrophobicity and distinct structural requirements in the hydrophobic region have important roles in signal peptide function.