Roles of Key Proline/Arginine Residues and Changes in Lipid Dynamics Reveal Interactions between the pH-Low Insertion Peptide (pHLIP) and Model Bilayers at Intermediate pH Values.

Abstract

The pH-Low Insertion Peptide (pHLIP) and its analogs sense the microenvironmental pH variations in tumorous cells and serve as useful anti-cancer drug deliveries. The pHLIP binds peripherally to membranes and adopts random coil conformation at the physiological pH. The peptide switches from random coil to α-helical conformation and inserts unidirectionally into membrane bilayers when pH drops below a critical transition value that has been routinely determined by the Trp fluorescence spectroscopy. Recent high-resolution studies using solid-state nuclear magnetic resonance spectroscopy (ssNMR) revealed the presence of thermodynamically stable intermediate states of membrane-associated pHLIP around the fluorescence-based transition pH value. However, the molecular structural features and their mechanistic roles of these intermediate states in the pH-driven membrane insertion process of pHLIP remain largely unknown. The present work utilizes ssNMR spectroscopy to explore (1) the mechanistic roles of key Proline and Arginine residues within the pHLIP sequence at intermediate pH values and (2) the changes in lipid dynamics at intermediate pH values in multiple types of model bilayers with anionic phospholipid and/or cholesterol. Our results demonstrate several molecular structural and dynamics changes at around the transition pH values, including the isomerization of proline-threonine backbone configuration, breaking of arginine-aspartic acid salt bridge and the formation of arginine-lipid interactions, and an universal decreasing of dynamics in lipid headgroups and alkyl chains. Overall, the outcomes provide important insights on the molecular interactions between pHLIP and membrane bilayers at intermediate pH values, and therefore prompt the understanding of pH-driven membrane insertion process of this anti-cancer drug-delivering peptide.