Kinetics of pHLIP peptide insertion into and exit from a membrane

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.

Ca2+‐dependent interactions between lipids and the tumor‐targeting peptide pHLIP

“…The transmembrane insertion of pHLIP into large unilamellar vesicles (LUV) was determined using Trp fluorescence, taking advantage of its two native Trp residues. The extensive use of Trp fluorescence in pHLIP studies provides well‐established benchmarks for its two membrane‐associated states (interfacial and transmembrane) in different lipid compositions 23–26,32 . In the case of POPS‐containing membranes, the interfacial form of pHLIP is characterized by a Trp λ Max ~357 nm, while its transmembrane form has a Trp λ Max ~343 nm 23,24 .…”

“…The extensive use of Trp fluorescence in pHLIP studies provides well-established benchmarks for its two membrane-associated states (interfacial and transmembrane) in different lipid compositions. [23][24][25][26]32 In the case of POPS-containing membranes, the interfacial form of pHLIP is characterized by a Trp λ Max $357 nm, while its transmembrane form has a Trp λ Max $343 nm. 23,24 In the absence of divalent cations, the addition of 3:1 POPC:POPS LUVs at pH 10 produced a Trp λ Max $356 nm, consistent with its interfacial state (Figure 2a, green).…”

Using Simulation to Understand the Role of Titration on the Stability of a Peptide–Lipid Bilayer Complex

“…Conventional fluorescence and CD spectroscopy techniques led to the initial suggestion that pHLIP can reversibly interconvert between states I, II, and III upon a transition from a neutral pH to an acidic, membrane-bound environment. , Subsequent studies have shown that this mechanism is much more nuanced. Kinetics studies indicated that multiple substates exist, with distinct pathways for insertion and exit. ,, Solid-state NMR and fluorescence spectroscopy revealed that each of the acidic residues in pHLIP possesses a unique p K a , with titration occurring in a nonlinear fashion, , as well as revealing that pHLIP exists in multiple states at slightly acidic pH. , In addition to these mechanistic studies, it has become clear that the function of pHLIP can be influenced by both the membrane environment and peptide composition. Non-PC lipids can prevent partitioning of pHLIP to form a stable membrane-bound complex; − physiological salt concentrations can decrease the propensity for pHLIP to insert into a membrane; even shifting the location of acidic residues in pHLIP or substituting acidic residues with more potent non-natural amino acids can enhance the effectiveness of insertion. , How do our results provide additional insights into this seemingly simple, yet complex mechanism?…”

“…Kinetics studies indicated that multiple substates exist, with distinct pathways for insertion and exit. 5,8,44 Solid-state NMR and fluorescence spectroscopy revealed that each of the acidic residues in pHLIP possesses a unique pK a , with titration occurring in a nonlinear fashion, 6,7 as well as revealing that pHLIP exists in multiple states at slightly acidic pH. 6,13 In addition to these mechanistic studies, it has become clear that the function of pHLIP can be influenced by both the membrane environment and peptide composition.…”

Is It Possible to Find an Antimicrobial Peptide That Passes the Membrane Bilayer with Minimal Force Resistance? An Attempt at a Predictive Approach by Molecular Dynamics Simulation

“…There is still no answer to the question about the mechanism of penetration of antimicrobial peptides (AMPs) through the bilayer of a membrane. Currently, work continues on the study of the penetration of peptides through the membrane [ 4 ]. The incorporation of the peptide into the lipid bilayer is triggered by a drop in pH, and its release from the bilayer is triggered by an increase in pH.…”