Pawlak M, Meseth U, Dhanapal B, Mutter M, Vogel H
Institute of Physical Chemistry IV, Swiss Federal Institute of Technology Lausanne, Lausanne.
Protein Sci. 1994 Oct;3(10):1788-805. doi: 10.1002/pro.5560031019.
Template-assembled proteins (TASPs) comprising 4 peptide blocks, each of either the natural melittin sequence (melittin-TASP) or of a truncated melittin sequence (amino acids 6-26, melittin6-26-TASP), C-terminally linked to a (linear or cyclic) 10-amino acid template were synthesized and characterized, structurally by CD, by fluorescence spectroscopy, and by monolayer experiments, and functionally, by electrical conductance measurements on planar bilayers and release experiments on dye-loaded vesicles. Melittin-TASP and the truncated analogue preferentially adopt alpha-helical structures in methanol (56% and 52%, respectively) as in lipid membranes. Unlike in methanol, the melittin-TASP self-aggregates in water. On an air-water interface, the differently sized molecules can be self-assembled and compressed to a compact structure with a molecular area of around 600 A2, compatible with a 4-helix bundle preferentially oriented perpendicular to the interface. The proteins reveal a strong affinity for lipid membranes. A partition coefficient of 1.5 x 10(9) M-1 was evaluated from changes of the Trp fluorescence spectra of the TASP in water and in the lipid bilayer. In planar lipid bilayers, TASP molecules are able to form defined ion channels, exhibiting a small single-channel conductance of 7 pS (in 1 M NaCl). With increasing protein concentration in the lipid bilayer, additional, larger conductance states of up to 1 nS were observed. These states are likely to be formed by aggregated TASP structures as inferred from a strongly voltage-dependent channel activity on membranes of large area. In this respect, melittin-TASP reveals channel features of the native peptide, but with a considerably lower variation in the size of the channel states. Compared to the free peptide, template-assembled melittin has a much higher membrane activity: it is about 100 times more effective in channel formation and 20 times more effective in releasing dye molecules from lipid vesicles. This demonstrates that the lytic properties are not solely related to channel formation.
合成了由4个肽块组成的模板组装蛋白（TASP），每个肽块要么是天然蜂毒素序列（蜂毒素-TASP），要么是截短的蜂毒素序列（氨基酸6 - 26，蜂毒素6 - 26 - TASP），其C端连接到一个（线性或环状）10氨基酸模板，并通过圆二色光谱（CD）、荧光光谱和单层实验进行结构表征，通过平面双层膜的电导测量和载有染料的囊泡的释放实验进行功能表征。蜂毒素-TASP和截短类似物在甲醇中（分别为56%和52%）与在脂质膜中一样优先采用α-螺旋结构。与在甲醇中不同，蜂毒素-TASP在水中会自聚集。在气-水界面上，不同大小的分子可以自组装并压缩成紧密结构，分子面积约为600 Ų，与优先垂直于界面定向的4螺旋束兼容。这些蛋白质对脂质膜表现出很强的亲和力。根据TASP在水和脂质双层中的色氨酸荧光光谱变化评估出分配系数为1.5×10⁹ M⁻¹。在平面脂质双层中，TASP分子能够形成明确的离子通道，在1 M NaCl中表现出7 pS的小单通道电导。随着脂质双层中蛋白质浓度的增加，观察到高达1 nS的额外更大电导状态。从大面积膜上强烈的电压依赖性通道活性推断，这些状态可能由聚集的TASP结构形成。在这方面，蜂毒素-TASP显示出天然肽的通道特征，但通道状态大小的变化要小得多。与游离肽相比，模板组装的蜂毒素具有更高的膜活性：它在通道形成方面效率高约100倍，在从脂质囊泡中释放染料分子方面效率高20倍。这表明裂解特性不仅仅与通道形成有关。
