Tiriveedhi Venkataswarup, Butko Peter
Department of Chemistry and Biochemistry, University of Southern Mississippi, Hattiesburg, Mississippi 39406, USA.
Biochemistry. 2007 Mar 27;46(12):3888-95. doi: 10.1021/bi602527t. Epub 2007 Mar 6.
Protein-transduction domains (PTDs) have been shown to translocate into and through the living cells in a rapid manner by an as yet unknown mechanism. Regardless of the mechanism of translocation, the first necessary step must be binding of the PTD peptide to the surface of the lipid membrane. We used fluorescence spectroscopy to study the interaction between PTD of the HIV-1 Tat protein (TAT-PTD; residues 47-60 of Tat, fluorescently labeled with tryptophan) and the lipid bilayer labeled with various fluorescence membrane probes. The TAT-PTD tryptophan exhibited a decrease in fluorescence intensity and an increase in anisotropy upon interaction with lipid bilayers. The fluorescence changes were linearly proportional to the density of negative charge in the membrane. Kinetic analysis of the interaction showed two apparent dissociation constants. The value of one dissociation constant (Kd1 = 2.6 +/- 0.6 microM), which accounted for 24% of the interaction, was found to be independent of the negative charge density, suggesting its nonelectrostatic nature. The value of the second dissociation constant (Kd2), which accounted for 76% of the interaction, decreased linearly from 610 +/- 150 to 130 +/- 30 microM with an increase in negative charge density from 0 to 25 mol %, suggesting this interaction is electrostatic in nature. Even though the binding was predominantly electrostatic, it could not be reversed by high salt, indicating the presence of a second, irreversible, step in the interaction with lipid. When TAT-PTD was bound to lipid vesicles labeled with 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH), fluorescence resonance energy transfer between the tryptophan and the probe occurred at a distance of 3.4 nm. No change in fluorescence anisotropy of either TMA-DPH or DPH was observed upon the interaction with TAT-PTD, indicating no significant disruption or perturbation of the lipid bilayer by the peptide. TAT-PTD did not cause dissipation of membrane potential (165 mV, negative inside). Inclusion of 3% pyrene-labeled phosphatidylglycerol (pyrene-PG) in the membrane revealed that TAT-PTD preferentially bound to the membrane in the liquid state. We conclude that membrane fluidity is an important physicochemical parameter, which may regulate binding of TAT-PTD to the membrane.
蛋白质转导结构域（PTDs）已被证明能通过一种尚不清楚的机制快速转运进入活细胞并穿过活细胞。无论转运机制如何，第一步必然是PTD肽与脂质膜表面结合。我们使用荧光光谱法研究了HIV-1 Tat蛋白的PTD（TAT-PTD；Tat的47-60位残基，用色氨酸进行荧光标记）与用各种荧光膜探针标记的脂质双层之间的相互作用。TAT-PTD色氨酸与脂质双层相互作用时，荧光强度降低，各向异性增加。荧光变化与膜中负电荷密度呈线性比例关系。相互作用的动力学分析显示有两个表观解离常数。其中一个解离常数的值（Kd1 = 2.6 +/- 0.6 microM），占相互作用的24%，发现其与负电荷密度无关，表明其非静电性质。第二个解离常数（Kd2）的值，占相互作用的76%，随着负电荷密度从0增加到25 mol%，从610 +/- 150 microM线性下降到130 +/- 30 microM，表明这种相互作用本质上是静电作用。尽管结合主要是静电作用，但高盐不能使其逆转，这表明在与脂质的相互作用中存在第二个不可逆步骤。当TAT-PTD与用1-(4-三甲基铵苯基)-6-苯基-1,3,5-己三烯（TMA-DPH）标记的脂质囊泡结合时，色氨酸与探针之间发生了距离为3.4 nm的荧光共振能量转移。与TAT-PTD相互作用时，未观察到TMA-DPH或DPH的荧光各向异性发生变化，这表明该肽对脂质双层没有明显的破坏或扰动。TAT-PTD不会导致膜电位（165 mV，内侧为负）的消散。在膜中加入3%的芘标记磷脂酰甘油（芘-PG）表明，TAT-PTD优先与液态膜结合。我们得出结论，膜流动性是一个重要的物理化学参数，可能调节TAT-PTD与膜的结合。
