Roles of Carboxyl Groups in the Transmembrane Insertion of Peptides

Abstract

We have used the pHLIP® peptide to study the roles of carboxyl groups in transmembrane peptide insertion. The pH (low) insertion peptide (pHLIP) binds to the surface of a lipid bilayer as a disordered peptide at neutral pH, and when the pH is lowered it inserts across the membrane to form a transmembrane helix. Peptide insertion is reversed when the pH is raised above the characteristic pKa (6.0). A key event facilitating the membrane insertion is the protonation of aspartic (Asp) and/or glutamic (Glu) acid residues, since at neutral pH their negatively charged side chains hinder membrane insertion. In order to gain mechanistic understanding, we studied the membrane insertion and exit of a series of pHLIP variants where the four Asp residues were sequentially mutated to nonacidic residues, including histidine (His). Our results show that the presence of His residues does not prevent the pH-dependent peptide membrane insertion at ~pH 4 driven by the protonation of carboxyl groups at the inserting end of the peptide. A further pH drop leads to the protonation of His residues in the TM part of peptide, which induces peptide exit from the bilayer. We also find that the number of ionizable residues that undergo a change in protonation during membrane insertion correlates with the pH-dependent insertion into and exit from the lipid bilayer, and that cooperativity increases with their number. We expect that our understanding will be used to improve the targeting of acidic diseased tissue by pHLIP peptides.

Membrane-Induced pKa Shifts in wt-pHLIP and Its L16H Variant

“…the peptide withdrawal from the membrane now occurs at lower pH values when the residue is ionized. 70 Hence, with a combination of these two strategies, we can design a peptide with two transitions, each having its own p K exp value. In this case, provided that the $\text{P}{K}_{\text{a}}^{\text{memb}}$ of the cationic group is lower than that of the anionic, the peptide will be stabilized in its membrane-inserted state in the pH range within the two $\text{P}{K}_{\text{a}}^{\text{memb}}$ values (Figure 4).…”

Membrane physical properties influence transmembrane helix formation

“…The k c parameter has been reported to modulate the coupled membrane attachment and insertion of the bacterial β-barrel porin OmpA (35). For pHLIP, both processes are independent, since after membrane attachment, insertion cannot proceed until key acidic groups in pHLIP are protonated (36). We therefore propose that k c influences pHLIP's state II but not state III.…”

“…Peptide synthesis, liposome preparation, analytical ultracentrifugation, fluorescence, and CD spectroscopy protocol are described elsewhere (36). A detailed description of the experimental methods is available in the SI Text.…”

Pore-forming Activity of the Escherichia coli Type III Secretion System Protein EspD

“…It is possible the insertion of EspD proceeds via a toroidal pore-forming mechanism that is known to be influenced by lipid composition (67) and may explain the requirement of anionic phospholipids for pore formation. It is conjectured that under acidic conditions the key amino acids on EspD become protonated, which lowers the thermodynamic barrier associated with insertion of charged residues through the hydrophobic core of the lipid bilayer (68,69). It is interesting to note that in the presence of high salt the dye release from LUVs was dramatically diminished, which FIGURE 9.…”