Protein Transduction Domains of HIV-1 and SIV TAT Interact with Charged Lipid Vesicles. Binding Mechanism and Thermodynamic Analysis.

Cell-penetrating peptides (CPPs) traverse cell membranes of cultured cells very efficiently by a mechanism not yet identified. Recent theories for the translocation suggest either the binding of the CPPs to extracellular glycosaminoglycans or the formation of inverted micelles with negatively charged lipids. In the present study, the binding of the protein transduction domains (PTD) of human (HIV-1) and simian immunodeficiency virus (SIV) TAT peptide (amino acid residues 47-57, electric charge z[sub p] = +8) to membranes containing various proportions of negatively charged lipid (POPG) is characterized. Monolayer expansion measurements demonstrate that TAT-PTD insertion between lipids requires loosely packed monolayer films. For densely packed monolayers (π > 29 mN/m) and lipid bilayers, no insertion is possible, and binding occurs via electrostatic adsorption to the membrane surface. Light scattering experiments show an aggregation of anionic lipid vesicles when the electric surface charge is neutralized by TAT-PTD, the observed stoichiometry being close to the theoretical value of 1:8. Membrane binding was quantitated with isothermal titration calorimetry and three further methods. The reaction enthalpy is ΔH[sup º] ≈ -1.5 kcal/mol peptide and is almost temperature-independent with ΔC[sub p, sup º] ∼0 kcal/(mol K), indicating equal contributions of polar and hydrophobic interactions to the reaction heat capacity. The binding of TAT-PTD to the anionic membrane is described by an electrostatic attraction/chemical partition model. The electrostatic attraction energy, calculated with the Gouy-Chapman theory, accounts for ∼80% of the binding energy. The overall binding constant, K[sub app], is ∼10³ - 10[sup 4] M[sup -1]. The intrinsic binding constant (K[sub p]), corrected for electrostatic effects and describing the partitioning of the peptide between the lipidwater interface and the membrane, is small and is K[sub... [ABSTRACT FROM AUTHOR]