Intramolecular interactions play key role in stabilization of pHLIP at acidic conditions

The pH‐Low Insertion Peptide (pHLIP) is a membrane‐active peptide that spontaneously folds into a transmembrane α ‐helix upon acidification. This activity enables pHLIP to potentially act as a vector for drugs related to diseases characterized by acidosis such as cancer or heart ischemia. Presently, due to aggregation‐based effects, formulations of pHLIP are only viable at near‐μM concentrations. In addition, since most of pHLIP's measurable qualities involve a membrane, probing the details of pHLIP in the interstitial region is difficult. In attempts to shed light on these issues, we performed constant pH molecular dynamics simulations on pHLIP as well as P20G, a variant with increased helicity, in solution at 0 and 150 mM NaCl over a broad range of pHs. In general, the addition of ions reduced the effective pK a of the acidic residues in pHLIP. P20G exhibits a higher helicity than pHLIP in general and is more compact than pHLIP at pH values under 4. In terms of charge effects, sodium cations localized predominantly to the C‐terminus of the peptide with a high density of acidic residues. Additionally, the salt bridge between R11 and D14 is by far the most favored and particularly so with pHLIP at 150 mM NaCl. We expect that this approach will be a valuable tool to screen variants of pHLIP for favorable properties in solution, an aspect of pHLIP design that to this point has largely been neglected.