Modulation of the pHLIP transmembrane helix insertion pathway.

Area of Science:

• Biophysics
• Membrane protein folding
• Peptide-lipid interactions

Background:

• The pH (low) insertion peptide (pHLIP) undergoes pH-dependent membrane insertion.
• Understanding sequence effects on pHLIP folding and insertion is crucial for biomolecular delivery.

Purpose of the Study:

• To investigate how sequence variations in pHLIP, including charged residues and attached cargoes, influence its membrane insertion kinetics and pathway.
• To explore the role of intermediate states in the folding/unfolding and insertion/exit processes of pHLIP variants.
• To propose a model for membrane-associated pHLIP folding and unfolding.

Main Methods:

• Steady-state and kinetic investigations of pHLIP variants.
• Utilizing variants with differing numbers of charged residues, attached polar cargoes, and single-Trp substitutions.
• Studying pH-dependent interactions with lipid bilayers.

Main Results:

• pHLIP variants show pH-dependent bilayer interactions.
• The number of charged residues correlates with insertion time and intermediate states, but not final helical structure.
• Polar cargoes introduce intermediate states and decrease insertion rates, with polarity correlating to the rate reduction.
• Simple pHLIP variants without charged or polar groups exhibit all-or-none folding/unfolding.

Conclusions:

• Intermediate states are not mandatory for pHLIP folding/unfolding.
• Sequence modifications, particularly cargo attachment, significantly impact pHLIP insertion kinetics and pathway.
• Findings inform the design of novel delivery agents for translocating molecules across cellular membranes.