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pH-low-insertion-peptide (pHLIP), a 36 residue peptide derived from the bacteriorhodopsin C-helix, can insert into a membrane at acidic pH to form a stable transmembrane α-helix. Such a unique property has shown promising applications for selective delivery of drugs to cancer tumors based on the physiological difference between healthy tissue and tumors. While healthy tissues have an extracellular pH (pHe) of 7.1-7.5, most solid tumors have a pHe of 6.5-7.0. pHLIP is believed to insert into a membrane through the protonation of Aspartic Acid residues located at positions 14,25,31,33. Wild type pHLIP completes insertion at a pH of ~6.1, so in order to increase insertion pH, four pHLIP variants (D31E - D31E,D33E - D31E,D33E,D25E - D31E,D33E,D25E,D14E) were synthesized with changes of Aspartic acid (D) residue to Glutamic Acid (E) residue at positions 14, 25, 31, 33. Glutamic Acid was chosen because it has a higher pKa than Aspartic Acid, and thus should increase the pH of pHLIP insertion to closer match pHe of tumors. The goal of this project is to use biophysical assays to study the interaction of new pHLIP variants with membranes and ultimately improve pHLIP’s performance as a pH sensor. Our data showed that all of four new variants exhibit higher insertion pH value than wild-type pHLIP, and the variant of D31E/D33E/D25E/D14E is the best with highest insertion pH of 6.4. Such findings will guide us for the future project of peptide-drug conjugation.



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Biophysical Characterization of Membrane Active pHLIP Peptides with Higher Insertion pH