Rao Yi, Kwok Sheldon J J, Lombardi Julien, Turro Nicholas J, Eisenthal Kenneth B
Department of Chemistry, Columbia University, New York, NY 10027
Department of Chemistry, Columbia University, New York, NY 10027.
Proc Natl Acad Sci U S A. 2014 Sep 2;111(35):12684-8. doi: 10.1073/pnas.1411817111. Epub 2014 Aug 18.
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.
转导激活蛋白（TAT）在艾滋病进展中起关键作用。研究表明，该蛋白中一个带 +8 电荷的氨基酸序列，即 TAT 肽，能使 TAT 蛋白穿透细胞膜。为探究 TAT 肽与细胞结合及转运的机制，研究人员使用磷脂脂质体作为细胞膜模拟物。我们采用表面电位敏感二次谐波产生（SHG）方法，这是一种无标记且具有界面选择性的方法，来研究 TAT 与阴离子型 1 - 棕榈酰 - 2 - 油酰 - sn - 甘油 - 3 - 磷酸 - 1'- rac - 甘油（POPG）和中性 1 - 棕榈酰 - 2 - 油酰 - sn - 甘油 - 3 - 磷酸胆碱（POPC）脂质体的结合。正是 SHG 对带电界面产生的静电场的敏感性使我们能够获得界面静电势。SHG 与泊松 - 玻尔兹曼方程共同得出表面电位对吸附 TAT 密度的依赖性。我们获得了 TAT 与 POPC 和 POPG 脂质体结合的解离常数 Kd 以及可结合到给定脂质体表面的 TAT 的最大数量。发现 POPC 的 Kd 为 7.5 ± 2 μM，POPG 的 Kd 为 29.0 ± 4.0 μM。当向脂质体溶液中加入 TAT 时，POPC 表面电位从 0 mV 变为 +37 mV，而对于 POPG，它从 -57 mV 变为 -37 mV。对 Kd 的数值计算，包括将泊松 - 玻尔兹曼方程应用于 TAT 脂质体 SHG 数据所获得的所有项，结果显示与近似解吻合良好。
