Interaction of synthetic HA2 influenza fusion peptide analog with model membranes

The interaction of the synthetic 21 amino acid peptide (AcE4K) with 1 -oleo yl-2-[caproyl-7-NBD]-sn-glycero-3-phosphocholine membranes is used as a mod el system for the pH-sensitive binding of fusion peptides to membranes. The sequence of AcE4K (Ac-GLFEAIAGFIENGWEGMIDGK) is based on the sequence of t he hemagglutinin HA2 fusion peptide and has similar partitioning into phosp hatidylcholine membranes as the viral peptide, pH-dependent partitioning in the membrane, circular dichroism, tryptophan fluorescence, change of membr ane area, and membrane strength, are measured to characterize various key a spects of the peptide-membrane interaction. The experimental results show t hat the partitioning of AcE4K in the membrane is pH dependent. The bound pe ptide inserts in the membrane, which increases the overall membrane area in a pH-dependent manner, however the depth of insertion of the peptide in th e membrane is independent of pH. This result suggests that the binding of t he peptide to the membrane is driven by the protonation of its three glutam atic acids and the aspartic acid, which results in an increase of the numbe r of bound molecules as the pH decreases from pH 7 to 4.5. The transition b etween the bound state and the free state is characterized by the Gibbs ene rgy for peptide binding. This Gibbs energy for pH 5 is equal to -30.2 kJ/mo l (-7.2 kcal/mol). Most of the change of the Gibbs energy during the bindin g of AcE4K is due to the enthalpy of binding -27.3 kJ/mol (-6.5 kcal/mol), while the entropy change is relatively small and is on the order of 6.4 J/m ol(.)K (2.3 cal/mol(.)K). The energy barrier separating the bound and the f ree state, is characterized by the Gibbs energy of the transition state for peptide adsorption. This Gibbs energy is equal to 51.3 kJ/mol (12.3 kcal/m ol). The insertion of the peptide into the membrane is coupled with work fo r creation of a vacancy for the peptide in the membrane. This work is calcu lated from the measured area occupied by a single peptide molecule (220 Ang strom (2)) and the membrane elasticity (190 mN/m), and is equal to 15.5 kJ/ mol (3.7 kcar/mol). The comparison of the work for creating a vacancy and t he Gibbs energy of the transition state shows that the work for creating a vacancy may have significant effect on the rate of peptide insertion and th erefore plays an important role in peptide binding. Because the work for cr eating a vacancy depends on membrane elasticity and the elasticity of the m embrane is dependent on membrane composition, this provides a tool for modu lating the pH for membrane instability by changing membrane composition. Th e insertion of the peptide in the membrane does not affect the membrane per meability for water, which shows that the peptide does not perturb substant ially the packing of the hydrocarbon region. However, the ability of the me mbrane to retain solutes in the presence of peptide is compromised, suggest ing that the inserted peptide promotes formation of short living pores. The integrity of the membrane is substantially compromised below pH 4.8 (thres hold pH), when large pores are formed and the membrane breaks down. The bin ding of the peptide in the pore region is reversible, and the pore size var ies on the experimental conditions, which suggests that the peptide in the pore region does not form oligomers.