"Mechanistic Studies of the Effect of pH-Dependent Peptide-Membrane Interactions on Conformational Switching"
Membrane proteins undergo conformational changes in response to a variety of external stimuli, driving crucial cellular processes. The pH Low Insertion Peptide (pHLIP) is a conformationally switching peptide with the unique ability to insert into cell membranes in acidic environments, making it a valuable tool for selective targeting and drug delivery to diseased acidic tissues, such as tumors. Peptide sequence, elevated levels of anionic lipids on the surface of cancer cells, and Ca2+ in the tumor environment have been shown to modulate pHLIP insertion. However, the mechanistic details and interplay of these three factors remain poorly understood. Understanding how these factors affect pHLIP membrane insertion will in turn inform our understanding of other pH-dependent membrane systems. To do this, systematic examinations of three pHLIP variants binding and insertion pH (pH50), as well as changes in secondary structure were measured across increasing phosphatidylserine (PS) content and [Ca2+], using tryptophan fluorescence and circular dichroism, respectively. Current results suggest that as PS content increases, pH50 decreases. Also, increasing Ca2+ increases pHLIP membrane binding, pH50 and helical content. Building off this work, high-resolution NMR using a 13C/15N labeled pHLIP peptide is used to resolve site specific peptide-lipid interactions, which requires a large quantity of peptide. Therefore, initial efforts have focused on optimization of the protein expression and purification protocols through changes in fusion protein tags and expression conditions. Furthering our understanding of how peptide sequence, divalent cations, and lipid composition affects pHLIP membrane insertion and site specific interactions will provide a path to better understanding other pH-dependent systems and to better tailor anti-tumor treatments to specific tumor environments.