pH (low) insertion peptide (pHLIP) inserts across a lipid bilayer as a helix and exits by a different path

Abstract

What are the molecular events that occur when a peptide inserts across a membrane or exits from it? Using the pH-triggered insertion of the pH low insertion peptide to enable kinetic analysis, we show that insertion occurs in several steps, with rapid (0.1 sec) interfacial helix formation, followed by a much slower (100 sec) insertion pathway to give a transmembrane helix. The reverse process of unfolding and peptide exit from the bilayer core, which can be induced by a rapid rise of the pH from acidic to basic, proceeds approximately 400 times faster than folding/insertion and through different intermediate states. In the exit pathway, the helix-coil transition is initiated while the polypeptide is still inside the membrane. The peptide starts to exit when about 30% of the helix is unfolded, and continues a rapid exit as it unfolds inside the membrane. These insights may guide understanding of membrane protein folding/unfolding and the design of medically useful peptides for imaging and drug delivery.folding kinetics | helix formation | lipid-peptide interaction | membranes | transmembrane helix

pHLIP peptide targets nanogold particles to tumors

“…A pHLIP is triggered by acidity to fold and insert across a membrane to form a stable transmembrane alpha helix (7). At neutral pH, pHLIP is in equilibrium between soluble and membrane-bound unstructured forms, whereas in a low-pH environment, the protonation of negatively charged residues (Asp or Glu) (8, 9) enhances peptide hydrophobicity, increasing the affinity of the peptide for the lipid bilayer and triggering peptide folding and subsequent membrane insertion (8,10). The Gibbs free energy of pHLIP binding to a 1-Palmitoyl-2-Oleoyl-snGlycero-3-Phosphocholine (POPC) from Avanti Polar Lipids liposome surface at 37°C is about -7 kcal/mol near neutral pH, and the additional free energy of folding and insertion across a lipid bilayer at low pH is nearly -2 kcal/mol (11).…”

“…The Gibbs free energy of pHLIP binding to a 1-Palmitoyl-2-Oleoyl-snGlycero-3-Phosphocholine (POPC) from Avanti Polar Lipids liposome surface at 37°C is about -7 kcal/mol near neutral pH, and the additional free energy of folding and insertion across a lipid bilayer at low pH is nearly -2 kcal/mol (11). Thus, the affinity of the peptide for a membrane at low pH is several times higher than at neutral pH, allowing pHLIP to distinguish and mark acidic diseased tissue (8,10,12,13). We have shown that the N terminus of pHLIP stays outside of the bilayer, whereas the C terminus inserts across the lipid bilayer at low pH (14,15).…”

pH-controlled delivery of luminescent europium coated nanoparticles into platelets

“…pHLIP has been shown to deliver cell impermeable compounds, such as phalloidin, and fluorescent compounds into cells by formation of a transmembrane helix upon change of the pH from physiological to 6.5 (15). This delivery is based on the ability of the pHLIP peptide, which loosely associates with the cell membrane at pH 7.4, to insert itself C-terminus first into the cell membrane upon a reduction in pH to ≤6.5 (16). The pHLIP mechanism of action has been determined in liposomes (17) and has been used in cells for the delivery of fluorescent molecules (18).…”

An in vitro study on the detection of ovarian cancer using a novel pH-specific targeting polypyrrole nanocontrasting agent.

“…In neutral or higher pH levels, the pHLIP is monomeric, remains non-reactive, and retains an unstructured or globular form. However, when placed in an acidic environment, it leads to the protonation of negatively charged residues on the helical segment and results in a shift in equilibrium, ultimately forming the alpha-helix structure [31]. Multiple studies have been successfully conducted with the use of pHLIPS as an additional modifier to existing polymer-based nanoparticles for pH-sensitive in vivo targeting for delivery as well as imaging [34,35].…”

“…To exploit the acidic extracellular pH as a targeting strategy for our polypyrrole nanocontrasting agent, we utilized a V7 pHLIP (pH Low Insertion Peptide), which changes conformation in response to its environment. In neutral pHe (7.2-7.4), it maintains a globular form and transforms to a transmembrane alpha-helix at acidic pHe conditions (6.4-6.8), this then allows the conjugated particle to insert and anchor into tumor cells [31][32][33].…”