Is the Bilayer Insertion of the Peptide KALP23 Under Kinetic or Thermodynamic Control?

Replacing a protein's sidechain can alter its interaction with a membrane, conferring a thermodynamic benefit or penalty. These thermodynamic consequences of mutations can be estimated using hydrophobicity scales, yet are challenging to validate experimentally. Here, we examine the structural and thermodynamic consequences of membrane interactions using two model peptides KALP23 and WALP23. In these peptides, respectively, two lysine residues or two tryptophan residues are placed at each end of a transmembrane helix. Application of several hydrophobicity scales suggests that the free energy of insertion of KALP23 is 10-15 kcal/mol less favorable than that of WALP23. According to these predictions, the lowest free energy for KALP would be in solution and not in the membrane, and KALP would be kinetically "trapped" in the transmembrane conformation during standard sample preparation based on co-solubilization with lipids in organic solvents. In order to quantitatively test this prediction, we utilized a different approach of sample preparation which utilizes fluorinated surfactants capable of maintain hydrophobic peptides in solution without interacting with the bilayer themselves. We demonstrated that both peptides can be inserted into the preformed vesicles, and that the free energy difference between their partitioning is < 1 kcal/mol. These results disprove the hypothesis that KALP peptides are kinetically trapped and open the doors for using this system for thermodynamic measurements to further test the additivity assumptions implicit in applications of hydrophobicity scales.