The Effect of Phosphatidylserine on a pH-Responsive Peptide Is Defined by Its Noninserting End

The acidity-triggered rational membrane (ATRAM) peptide was designed to target acidic diseases such as cancer. An acidic extracellular medium, such as that found in aggressive tumors, drives the protonation of the glutamic acids in ATRAM, leading to the membrane translocation of its C-terminus and the formation of a transmembrane helix. Compared to healthy cells, cancerous cells often increase exposure of the negatively charged phosphatidylserine (PS) on the outer leaflet of the plasma membrane. Here we use a reconstituted vesicle system to explore how PS influences the interaction of ATRAM with membranes. To explore this, we used two new variants of ATRAM, termed K2-ATRAM and Y-ATRAM, with small modifications at the noninserting N-terminus. We observed that the effect of PS on the membrane insertion pK and lipid partitioning hinged on the sequence of the noninserting end. Our data additionally indicate that the effect of PS on the insertion pK does not merely depend on electrostatics, but it is multifactorial. Here we show how small sequence changes can impact the interaction of a peptide with membranes of mixed lipid composition. These data illustrate how model studies using neutral bilayers, which do not mimic the negative charge found in the plasma membrane of cancer cells, may fail to capture important aspects of the interaction of anticancer peptides with tumor cells. This information can guide the design of therapeutic peptides that target the acidic environments of different diseased tissues.