Lipid Headgroups Modulate Membrane Insertion of pHLIP Peptide

Abstract

The pH low insertion peptide (pHLIP) is an important tool for drug delivery and visualization of acidic tissues produced by various maladies, including cancer, inflammation, and ischemia. Numerous studies indicate that pHLIP exists in three states: unfolded and soluble in water at neutral pH (State I), unfolded and bound to the surface of a phosphatidylcholine membrane at neutral pH (State II), and inserted across the membrane as an α-helix at low pH (State III). Here we report how changes in lipid composition modulate this insertion scheme. First, the presence of either anionic lipids, cholesterol, or phosphoethanolamine eliminates membrane binding at neutral pH (State II). Second, the apparent pKa for the insertion transition (State I → State III) is increased with increasing content of anionic lipids, suggesting that electrostatic interactions in the interfacial region modulate protonation of acidic residues of pHLIP responsible for transbilayer insertion. These findings indicate a possibility for triggering protonation-coupled conformational switching in proteins at membrane interfaces through changes in lipid composition.

Applications of pHLIP Technology for Cancer Imaging and Therapy

“…The inserted peptide leaves its N-terminus in the extracellular space while the C-terminus is translocated across the membrane into the cytoplasm. The lipid composition of the cell membrane can contribute to modulation the binding and insertion into the membrane of a pHLIP [20, 41, 42]. …”

Using membrane composition to fine-tune the pKa of an optical liposome pH sensor

“…28-30 Conversely, the interfacial electrostatic surface potential has been observed to drive changes in p K a for optical probes in micelles, 31 pH responsive spin labels in liposomes, 13, 32 and liposome insertion rates by transmembrane peptides. 33 To distinguish these possible explanations, the pH titration experiments were repeated using liposomes containing probe 2 and 20 mol % of POPS or the analogous anionic phospholipids, POPA, and POPG. ‡ As shown in Fig.…”

Cooperative Non-bonded Forces Control Membrane Binding of the pH-Low Insertion Peptide pHLIP

“…A more general issue we have begun to address is the complexity of how pHLIP interacts with a cell membrane. The field has progressed from the conventional three-state model to begin to account for differences in membrane composition from POPC (15)(16)(17) as well as utilizing advanced spectroscopic techniques that allow for a more detailed characterization of pHLIP conformational sampling in the bound and inserted states (13,14,50,51). A combination of experimental and computational approaches will be required to fully understand how pHLIP functions, which will subsequently enable design of more specific and effective variants of pHLIP.…”

“…When adding anionic phospholipids (i.e., phosphatidylserine) to the vesicles, the free energy of binding is essentially the same (15). However, Ladokhin and coworkers determined that state II is spectroscopically silent in the presence of non-PC lipids -pHLIP will bind to lipid vesicles but does not partition below the headgroup region, leading to a lack of change in tryptophan fluorescence from state I to state II (16,17). These more recent studies strongly suggest that a more complex mechanism of binding exists for pHLIP.…”