Alterations in the hydrophilic segment of the maltose-binding protein (MBP) signal peptide that affect either export or translation of MBP

Abstract

Mutations that reduce the net positive charge within the hydrophilic segments of the signal peptides of several prokaryotic exported proteins can result in a reduction in the rate of protein export, as well as a reduction in protein synthesis (M. N. Hall, J. Gabay, and M. Shwartz, EMBO J. 2:15-19, 1983 revealed that the role of the hydrophilic segment in the export process can be functionally separated from any role in translation. Taken together, these results strongly suggest that the hydrophilic segment of the MBP signal peptide is not involved in a mechanism that couples MBP translation to export and argue against the presence of a mechanism that obligatorily couples translation to protein export in Escherichia coli.Proteins destined for export in Escherichia coli are synthesized as precursor species with an amino-terminal extension termed the signal peptide that is responsible for initiating the export of proteins from the cytoplasm. The periplasmic maltose-binding protein (MBP) of E. coli, the product of the malE gene, is synthesized with a typical bacterial signal peptide that shares little primary amino acid homology with other signal peptides but does exhibit conserved features characteristic of signal peptides of both prokaryotic and eukaryotic origin. The first eight residues of the MBP signal peptide constitute the hydrophilic segment, a distinct region that carries a net positive charge due to the presence of three basic residues. The hydrophilic segment precedes the hydrophobic core, a region devoid of charged residues that is predicted to assume an a-helical conformation. Following the hydrophobic core is the cleavage site for signal peptidase I. (For a comprehensive review of the MBP signal peptide and MBP export, see reference 4.)A specific role for the basic residues of the hydrophilic segment in protein export was first suggested in the loop model (18). The basic residues were proposed to initiate an ionic interaction between the signal peptide and the nega-* Corresponding author.

Effect of alteration of charged residues at the N termini of signal peptides on protein export in Bacillus subtilis

“…However, Npr mutant D5N6 showed a significant inhibition of synthesis and Npr mutant D5D6 had a slightly enhanced level of synthesis. This effect on synthesis is probably related to alterations in the mRNA structure rather than an obligate mechanistic coupling of translation and secretion (20).…”

“…An extensive characterization of the N region of the maltose-binding protein signal peptide was carried out by Bassford's laboratory. Those studies revealed that (i) a net positive charge is not essential for the export of the maltose-binding protein (19), (ii) the effect of charged alteration is dependent on the adjacent hydrophobic core region of the signal peptide (21), and (iii) positively charged residues are not involved in coupling translation and export (20). The roles of positively charged residues in the export of staphylokinase and lipoprotein have also been studied (7,8).…”

In Silico Evaluation of Different Signal Peptides for the Secretory Production of Human Growth Hormone in E. coli

“…Substituting of the basic residues with neutral or acidic residues in the signal peptide sequence has various effects on the protein secretion, including a reduction in the rate of the protein synthesis and exportation (Inouye et al 1982;Nesmeyanova et al 1997), an alteration in the cleavage site of the signal peptide (Suominen et al 1995), or no significant effect on the protein secretion (Vlasuk et al 1983). It has been also believed that the presence of the basic residue, particularly lysine, at the second codon (P2) improves the rate of protein secretion (Puziss et al 1992). Therefore, the presence of one or more basic amino acids in the n-region is probably essential for the development of an applicable signal peptide (Low et al 2013).…”

Molecular Determinants for Protein Stabilization by Insertional Fusion to a Thermophilic Host Protein

“…The first five residues of PfMBP, which were numbered according to previous studies, originated from wild‐type E. coli maltodextrin‐binding protein (EcMBP), not from wild‐type PfMBP, and therefore were not included. Instead, the signal sequence of EcMBP (residue 1—30), which precedes the first residues of mature EcMBP (i.e., residue 31), was genetically added to the N terminus of PfMBP. The nucleic acid sequence encoding PC1 β‐lactamase (BLA PC1 ) from Staphylococcus aureus was synthesized by Operon Biotechnologies and supplied in the pCR 2.1‐TOPO plasmid vector.…”