Label-free probe of HIV-1 TAT peptide binding to mimetic membranes

The transacting activator of transduction (TAT) protein plays a key role in the progression of AIDS. Studies have shown that a +8 charged sequence of amino acids in the protein, called the TAT peptide, enables the TAT protein to penetrate cell membranes. To probe mechanisms of binding and translocation of the TAT peptide into the cell, investigators have used phospholipid liposomes as cell membrane mimics. We have used the method of surface potential sensitive second harmonic generation (SHG), which is a label-free and interface-selective method, to study the binding of TAT to anionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-1′-rac-glycerol (POPG) and neutral 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) liposomes. It is the SHG sensitivity to the electrostatic field generated by a charged interface that enabled us to obtain the interfacial electrostatic potential. SHG together with the Poisson–Boltzmann equation yielded the dependence of the surface potential on the density of adsorbed TAT. We obtained the dissociation constants K_d for TAT binding to POPC and POPG liposomes and the maximum number of TATs that can bind to a given liposome surface. For POPC K_d was found to be 7.5 ± 2 μM, and for POPG K_d was 29.0 ± 4.0 μM. As TAT was added to the liposome solution the POPC surface potential changed from 0 mV to +37 mV, and for POPG it changed from −57 mV to −37 mV. A numerical calculation of K_d, which included all terms obtained from application of the Poisson–Boltzmann equation to the TAT liposome SHG data, was shown to be in good agreement with an approximated solution.The HIV type 1 (HIV-1) transacting activator of transduction (TAT) is an important regulatory protein for viral gene expression (1–3). It has been established that the TAT protein has a key role in the progression of AIDS and is a potential target for anti-HIV vaccines (4). For the TAT protein to carry out its biological functions, it needs to be readily imported into the cell. Studies on the cellular internalization of TAT have led to the discovery of the TAT peptide, a highly cationic 11-aa region (protein transduction domain) of the 86-aa full-length protein that is responsible for the TAT protein translocating across phospholipid membranes (5–8). The TAT peptide is a member of a class of peptides called cell-penetrating peptides (CPPs) that have generated great interest for drug delivery applications (ref. 9 and references therein). The exact mechanism by which the TAT peptide enters cells is not fully understood, but it is likely to involve a combination of energy-independent penetration and endocytosis pathways (8, 10). The first step in the process is high-affinity binding of the peptide to phospholipids and other components on the cell surface such as proteins and glycosaminoglycans (1, 9).The binding of the TAT peptide to liposomes has been investigated using a variety of techniques, each of which has its own advantages and limitations. Among the techniques are isothermal titration calorimetry (9, 11), fluorescence spectroscopy (12, 13), FRET (12, 14), single-molecule fluorescence microscopy (15, 16), and solid-state NMR (17). Second harmonic generation (SHG), as an interface-selective technique (18–24), does not require a label, and because SHG is sensitive to the interface potential, it is an attractive method to selectively probe the binding of the highly charged (+8) TAT peptide to liposome surfaces. Although coherent SHG is forbidden in centrosymmetric and isotropic bulk media for reasons of symmetry, it can be generated by a centrosymmetric structure, e.g., a sphere, provided that the object is centrosymmetric over roughly the length scale of the optical coherence, which is a function of the particle size, the wavelength of the incident light, and the refractive indexes at ω and 2ω (25–30). As a second-order nonlinear optical technique SHG has symmetry restrictions such that coherent SHG is not generated by the randomly oriented molecules in the bulk liquid, but can be generated coherently by the much smaller population of oriented interfacial species bound to a particle or planar surfaces. As a consequence the SHG signal from the interface is not overwhelmed by SHG from the much larger populations in the bulk media (25–28).The total second harmonic electric field, E_2ω, originating from a charged interface in contact with water can be expressed as (31–33)E2ω∝∑iχc,i(2)EωEω+∑jχinc,j(2)EωEω+χH2O(3)EωEωΦ,1]where χc,i(2) represents the second-order susceptibility of the species i present at the interface; χinc,j(2) represents the incoherent contribution of the second-order susceptibility, arising from density and orientational fluctuations of the species j present in solution, often referred to as hyper-Rayleigh scattering; χH2O(3) is the third-order susceptibility originating chiefly from the polarization of the bulk water molecules polarized by the charged interface; Φ is the potential at the interface that is created by the surface charge; and E_ω is the electric field of the incident light at the fundamental frequency ω. The second-order susceptibility, χc,i(2), can be written as the product of the number of molecules, N, at the surface and the orientational ensemble average of the hyperpolarizability α_i⁽²⁾ of surface species i, yielding χc,i(2)=N〈αi(2)〉 (18). The bracket ?? indicates an orientational average over the interfacial molecules. The third term in Eq. 1 depicts a third-order process by which a second harmonic field is generated by a charged interface. This term is the focus of our work. The SHG signal is dependent on the surface potential created by the electrostatic field of the surface charges, often called the χ⁽³⁾ contribution to the SHG signal. The χ⁽³⁾ method has been used to extract the surface charge density of charged planar surfaces and microparticle surfaces, e.g., liposomes, polymer beads, and oil droplets in water (21, 25, 34–39).In this work, the χ⁽³⁾ SHG method is used to explore a biomedically relevant process. The binding of the highly cationic HIV-1 TAT peptide to liposome membranes changes the surface potential, thereby enabling the use of the χ⁽³⁾ method to study the binding process in a label-free manner. Two kinds of liposomes, neutral 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and anionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-1′-rac-glycerol (POPG), were investigated. The chemical structures of TAT, POPC, and POPG lipids are shown in Scheme 1.Open in a separate windowScheme 1.Chemical structures of HIV-1 TAT (47–57) peptide and the POPC and POPG lipids.