Modulation of the pHLIP Transmembrane Helix Insertion Pathway

Abstract

The membrane-associated folding/unfolding of pH (low) insertion peptide (pHLIP) provides an opportunity to study how sequence variations influence the kinetics and pathway of peptide insertion into bilayers. Here, we present the results of steady-state and kinetics investigations of several pHLIP variants with different numbers of charged residues, with attached polar cargoes at the peptide's membrane-inserting end, and with three single-Trp variants placed at the beginning, middle, and end of the transmembrane helix. Each pHLIP variant exhibits a pH-dependent interaction with a lipid bilayer. Although the number of protonatable residues at the inserting end does not affect the ultimate formation of helical structure across a membrane, it correlates with the time for peptide insertion, the number of intermediate states on the folding pathway, and the rates of unfolding and exit. The presence of polar cargoes at the peptide's inserting end leads to the appearance of intermediate states on the insertion pathway. Cargo polarity correlates with a decrease of the insertion rate. We conclude that the existence of intermediate states on the folding and unfolding pathways is not mandatory and, in the simple case of a polypeptide with a noncharged and nonpolar inserting end, the folding and unfolding appears as an all-or-none transition. We propose a model for membrane-associated insertion/folding and exit/unfolding and discuss the importance of these observations for the design of new delivery agents for direct translocation of polar therapeutic and diagnostic cargo molecules across cellular membranes.

Family of pH (low) insertion peptides for tumor targeting

“…The data are consistent with the view that the acetylated N terminus of Var14 inserts across the bilayer and the amidated C terminus stays outside, reversing the direction of insertion. The characteristic time of Var1-2 insertion, which has fewer protonatable residues at its C terminus than WT, was about 1-2 s, which correlates well with our recent findings (13). Further truncation of the peptideinserting end, which reduces the number of protonatable residues, resulted in faster peptide insertion, completed within first 30-100 ms for Var3-13.…”

“…We have found in recent work that the kinetics of insertion correlate with the number of protonatable groups at the inserting end or the presence of polar cargo (13). The characteristic membrane insertion time for WT-pHLIP is about 30 s, and slightly faster kinetics were observed for Var14, which is the reverse sequence of WT.…”

“…Oriented CD was measured from the supported bilayers deposited on a stack of quartz slides with special polish for far UV measurements as described previously (13).…”

Enhancement of radiation effect on cancer cells by gold-pHLIP

“…The delivery approach we propose is based on the energy of membrane-associated folding of peptides from the pHLIP family to target cellular membranes in a pHdependent manner (22,24,37). At pH < 7.0, pHLIPs insert into the lipid bilayer of the membrane, which is accompanied by a coil-helix transition and formation of a transmembrane helix.…”

pH (low) insertion peptide (pHLIP) targets ischemic myocardium

“…The insertion process is unidirectional and rapid (<2 min in lipid vesicles) (3)(4)(5). Variations of the pHLIP sequence can be used to change the time required for insertion across and exit from the membrane, ranging from milliseconds to minutes (6). The apparent pK of insertion across a liposomal bilayer membrane is 6.0 for the original WT-pHLIP sequence (2).…”