A PHOTOGENERATED PORE-FORMING PROTEIN
A PHOTOGENERATED PORE-FORMING PROTEIN
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DOI：
10.1016/1074-5521(95)90220-1
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发表时间：
1995-06-01
影响因子：
--
通讯作者：
BAYLEY, H
中科院分区：
文献类型：
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作者：
CHANG, CY;NIBLACK, B;BAYLEY, H
背景资料：用细菌成孔蛋白透化细胞是细胞生物学中的重要技术，其允许将小试剂交换到细胞的细胞质中。另一个值得注意的技术是使用笼状分子，其活性通过添加光可去除的保护基团来阻断。这允许在空间和时间控制的情况下在细胞上或细胞中光生试剂。在这里，我们联合收割机这些方法来产生一个笼状孔形成蛋白的控制permeabilizationofcells.Results：2-溴-2-（2-硝基苯基）乙酸（BNPA），一个水溶性的半胱氨酸定向试剂笼肽和蛋白质与α-羧基-2-硝基苄基（CNB）的保护基团，合成。通过在300 nm下照射，以高产率从γ-Glu-CysCNB-Gly释放谷氨酸（γ-Glu-Cys-Gly）。基于这一发现，扫描诱变被用来寻找一个单一的半胱氨酸突变体的孔形成蛋白葡萄球菌α-溶血素（α HL）适合笼。当α HL-R104 C用BNPA衍生化时，对兔红细胞的成孔活性丧失。近紫外线照射导致再生的半胱氨酸巯基和恢复的孔形成activity.Conclusions：笼孔形成蛋白是潜在的有用的透化一个细胞在一个集合的细胞或一个区域的质膜的一个单一的细胞。因此，α HL-R104 C-CNB和设计用于产生各种直径的孔的其他笼状蛋白质应该可用于许多目的。例如，将试剂引入网络的一个细胞或单个细胞的一个区域的能力可用于神经元调节的研究。此外，BNPA通常可用于笼化含半胱氨酸的肽和单半胱氨酸突变蛋白，以研究例如细胞信号传导或蛋白质的结构变化。
Background: The permeabilization of cells with bacterial pore-forming proteins is an important technique in cell biology that allows the exchange of small reagents into the cytoplasm of a cell. Another notable technology is the use of caged molecules whose activities are blocked by addition of photoremovable protecting groups. This allows the photogeneration of reagents on or in cells with spatial and temporal control. Here, we combine these approaches to produce a caged pore-forming protein for the controlled permeabilization of cells.Results: 2-Bromo-2-(2-nitrophenyl)acetic acid (BNPA), a water-soluble cysteine-directed reagent for caging peptides and proteins with the alpha-carboxy-2-nitrobenzyl (CNB) protecting group, was synthesized. Glutathione (gamma-Glu-Cys-Gly) was released in high yield from gamma-Glu-CysCNB-Gly by irradiation at 300 nm. Based on this finding, scanning mutagenesis was used to find a single-cysteine mutant of the pore-forming protein staphylococcal alpha-hemolysin (alpha HL) suitable for caging. When alpha HL-R104C was derivatized with BNPA, pore-forming activity toward rabbit erythrocytes was lost. Near UV irradiation led to regeneration of the cysteine sulfhydryl group and the restoration of pore-forming activity.Conclusions: Caged pore-forming proteins are potentially useful for permeabilizing one cell in a collection of cells or one region of the plasma membrane of a single cell. Therefore, alpha HL-R104C-CNB and other caged proteins designed to create pores of various diameters should be useful for many purposes. For example, the ability to introduce reagents into one cell of a network or into one region of a single cell could be used in studies of neuronal modulation. Further, BNPA should be generally useful for caging cysteine-containing peptides and single-cysteine mutant proteins to study, for example, cell signaling or structural changes in proteins.