title: Label-free probe of HIV-1 TAT peptide binding to mimetic membranes.
abstract: 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 Kd for TAT binding to POPC and POPG liposomes and the maximum number of TATs that can bind to a given liposome surface. For POPC Kd was found to be 7.5 ± 2 μM, and for POPG Kd 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 Kd, 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.
authors.affiliation: Department of Chemistry, Columbia University, New York, NY 10027 kbe1@columbia.edu yirao@temple.edu.
authors.forename: Yi
authors.initials: Y
authors.lastname: Rao
authors.affiliation: Department of Chemistry, Columbia University, New York, NY 10027.
authors.forename: Sheldon J J
authors.initials: SJ
authors.lastname: Kwok
authors.affiliation: Department of Chemistry, Columbia University, New York, NY 10027.
authors.forename: Julien
authors.initials: J
authors.lastname: Lombardi
authors.affiliation: Department of Chemistry, Columbia University, New York, NY 10027.
authors.forename: Nicholas J
authors.initials: NJ
authors.lastname: Turro
authors.affiliation: Department of Chemistry, Columbia University, New York, NY 10027 kbe1@columbia.edu yirao@temple.edu.
authors.forename: Kenneth B
authors.initials: KB
authors.lastname: Eisenthal
mesh_terms: D000163:Acquired Immunodeficiency Syndrome / Q000378:metabolism / Q000821:virology*; D003102:Colloids / Q000378:metabolism; D016503:Drug Delivery Systems; D015497:HIV-1 / Q000378:metabolism*; D006801:Humans; D007834:Lasers; D008081:Liposomes / Q000378:metabolism; D008567:Membranes, Artificial; D008956:Models, Chemical; D018716:Molecular Mimicry; D010743:Phospholipids / Q000378:metabolism; D011485:Protein Binding; D013059:Spectrum Analysis, Raman / Q000379:methods*; D013499:Surface Properties; D014867:Water / Q000378:metabolism; D054322:tat Gene Products, Human Immunodeficiency Virus / Q000378:metabolism*
chemical_list: D003102:Colloids; D008081:Liposomes; D008567:Membranes, Artificial; D010743:Phospholipids; D054322:tat Gene Products, Human Immunodeficiency Virus; D014867:Water
keywords: Gouy–Chapman model; biomolecules/water interface; colloid/water interface; hyper-Rayleigh scattering; nonlinear optical spectroscopy