Conformational Sampling of the pH Low Insertion Peptide is Tuned by pH

Abstract

The pH Low Insertion Peptide (pHLIP) is a 36 residue, pH-sensitive membrane-active peptide that is coiled both in solution and when bound to a membrane at physiological pH. Upon acidification, pHLIP folds into an α-helix and inserts unidirectionally into the bilayer. Thus, pHLIP could act as a potential vector for targeted drug delivery to or diagnostic imaging of tissues associated with acidosis, such as cancer or heart ischemia. However, without a detailed description of the biophysical interactions of pHLIP that lead to function, it will be difficult to design variants with increased targeting abilities. A recent study utilizing pH probes on cancerous cells has determined an increased radius of extracellular space is affected by acidosis [1], and therefore a peptide approaching the bilayer would experience an acidic environment before interacting with the membrane. With this in mind, we employed constant pH molecular dynamics (MD) simulations to determine potential changes in conformation of pHLIP as it transitions into the acidic extracellular space. Surprisingly, we found that a single amino acid residue, D31, has a noticeably higher pKa than what is expected from the experimentally determined pKa of aspartic acid residues. In addition, we observe formation of a stable helical fold from D14 to T18 at pH values below 4.5, suggesting pHLIP has the potential to sample conformations (i.e., α-helices) that facilitate partitioning into the membrane while still in solution. Our results provide a unique and essential perspective on the behavior of pHLIP as it transitions into acidic microenvironments such as the extracellular region of cancerous cells. [1] Luo (2018) Anal. Chem. 90:5803-5809.