Interfacial folding and membrane insertion of a designed helical peptide

Area of Science:

• Biochemistry
• Membrane Biophysics
• Protein Folding

Background:

• Nonconstitutive membrane proteins, like diphtheria toxin, require refolding at membrane interfaces for penetration.
• Understanding the energetics of interface-mediated insertion of model transmembrane helices is crucial.
• Hydrophobic peptides tend to aggregate in aqueous solutions, complicating studies.

Purpose of the Study:

• To design and characterize a model peptide (TMX-3) for studying membrane insertion energetics.
• To investigate the factors controlling peptide behavior in aqueous and membrane environments.
• To quantitatively analyze the thermodynamics of interface-mediated peptide partitioning.

Main Methods:

• Design of a 31-residue peptide (TMX-3) with a proline residue and two histidine residues.
• Kinetic analysis of peptide partitioning into membranes.
• Induction and measurement of peptide helicity.
• pH-dependent studies to analyze histidine pKa shifts.
• Fluorescence titration to determine free energies of partitioning.

Main Results:

• TMX-3 exhibited complex partitioning kinetics and induced helicity upon membrane interaction.
• Histidine residues shifted pKa values, allowing pH-controlled interactions.
• Two binding modes were observed: interfacial (IF) and partial transmembrane (TM) insertion.
• Free energies of IF partitioning were -6.7 kcal/mol (pH 7.6) and -5.0 kcal/mol (pH 4.5).

Conclusions:

• Histidine titration plays a significant role in pH-dependent refolding of toxins on membrane interfaces.
• The interfacial folded state is the most stable conformation for TMX-3 under studied conditions.
• Interfacial states are critical for the spontaneous refolding and insertion of diphtheria and other membrane toxins.