Abstract
To advance mechanistic understanding of membrane-associated peptide folding and insertion, we have studied the kinetics of three single tryptophan pHLIP (pH-Low Insertion Peptide) variants, where tryptophan residues are located near the N terminus, near the middle, and near the inserting C-terminal end of the pHLIP transmembrane helix. Single-tryptophan pHLIP variants allowed us to probe different parts of the peptide in the pathways of peptide insertion into the lipid bilayer (triggered by a pH drop) and peptide exit from the bilayer (triggered by a rise in pH). By using pH jumps of different magnitudes, we slowed down the processes and established the intermediates that helped us to understand the principles of insertion and exit. The obtained results should also aid the applications in medicine that are now entering the clinic.
Highlights
Introduction pHLow Insertion Peptides are being increasingly studied to gain insights concerning peptide folding and insertion into membranes, and to apply them in medicine.1 pHLIP® is a registered trademark owned by the Rhode Island Board of EducationBecause their membrane insertion from a water-soluble state is triggered by pH changes, a rich opportunity is created for chemical, kinetic and computational studies, as shown by the expanding literature from a growing number of laboratories and ongoing efforts to use them as medically useful acidity sensors in vivo
We studied the kinetics of insertion into and exit from the lipid bilayer of POPC
Liposomes using three single Trp pHLIP variants, with the Trp reporters positioned in each flanking region and in the membrane inserted region. pHLIP peptides with a single tryptophan residue allow a “clean” photophysical signals originating from single fluorophores, avoiding spectral heterogeneity. pHLIP variants were designed based on the closely related group of the WT sequences [24], which contains several protonatable residues at the membrane inserting Cterminal end of the peptide
Summary
Introduction pHLow Insertion Peptides (pHLIPs®1) are being increasingly studied to gain insights concerning peptide folding and insertion into membranes, and to apply them in medicine.1 pHLIP® is a registered trademark owned by the Rhode Island Board of EducationBecause their membrane insertion from a water-soluble state is triggered by pH changes, a rich opportunity is created for chemical, kinetic and computational studies, as shown by the expanding literature from a growing number of laboratories and ongoing efforts to use them as medically useful acidity sensors in vivo. Weak surface binding is useful for a pH-sensing agent, since the pH at the surfaces of cancer cells is 0.5-0.7 pH units lower than the bulk extracellular pH and independent of tumor (tissue) perfusion [19, 22]. Another feature of the pHLIP delivery system is that these peptides undergo a cooperative coilhelix transition in response to a pH change, and the pK and cooperativity of the transition are tunable by sequence variation. These parameters have utility for pHLIP applications to real biological systems
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