Interfacial folding and membrane insertion of a designed helical peptide.

Nonconstitutive membrane-active proteins, such as diphtheria toxin, must refold on membrane interfaces in the course of membrane penetration. A useful step in deciphering this process is to understand quantitatively the energetics of interface-mediated insertion of model transmembrane helices. A difficulty is that peptides that are sufficiently hydrophobic to span a lipid bilayer have a strong tendency to aggregate in the aqueous phase. To learn how to control the aqueous and membrane behavior of model peptides, we designed a 31-residue peptide (TMX-3) whose properties are described here. TMX-3 has two important structural features: a proline residue in the hydrophobic core that discourages the formation of highly helical aggregates in solution and two histidine residues that allow control of membrane and solution interactions by means of pH changes. The partitioning of TMX-3 into membranes followed complex kinetics, induced helicity, and shifted the histidine pK(a) from 6.8 to approximately 6. Topology measurements disclosed two general modes of TMX-3 binding: interfacial (IF) at low peptide concentrations and partial transmembrane (TM) insertion at higher concentrations. Both modes were reversible and, consequently, suitable for thermodynamic analysis. The free energies of IF partitioning of TMX-3 with deprotonated (pH 7.6) and protonated histidines (pH 4.5) were estimated by fluorescence titration to be -6.7 and -5.0 kcal/mol, respectively. These results show that histidine titration is likely to be important in the pH-dependent refolding of toxins on membrane interfaces and that the most favored state of TMX-3 under any conditions is the IF folded state, which emphasizes the importance of such states in the spontaneous refolding and insertion of diphtheria and other membrane toxins.