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首页> 外文期刊>Chemistry: A European journal >Construction of artificial signal transducers on a lectin surface by post-photoaffinity-labeling modification for fluorescent saccharide biosensors
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Construction of artificial signal transducers on a lectin surface by post-photoaffinity-labeling modification for fluorescent saccharide biosensors

机译：通过光亲和标记后修饰修饰荧光糖生物传感器在凝集素表面上构建人工信号传感器

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摘要

A new general method, post-photoaffinity-labeling modification (PPALM), for constructing fluorescent saccharide biosensors based on naturally occurring saccharide-binding proteins, lectins, is described in detail. An active-site-directed incorporation of a masked reactive site into a lectin was conducted by using a photoaffinity labeling technique followed by demasking and then chemical modification to yield a fluorescent lectin. Two photoaffinity labeling reagents were designed and synthesized in this study. The labeling reagent with a photoreactive site appended through a disulfide link to a mannoside unit was bound to the saccharide-binding pocket of the lectin concanavalin A (ConA). After light irradiation, the mannoside unit was cleaved by reduction. The unique thiol group thus produced was site-specifically modified with various fluorescent groups (dansyl, counmarin, or dimethylaminobenzoate derivatives) to afford fluorescent Con As. The labeling site was characterized by protease-catalyzed digestion followed by HPLC, MALDI-TOF MS, and tandem mass-mass spectrometry; these methods indicated that the photolabeling step is remarkably site specific. Strong fluorescence was observed in the engineered Con A with a fluorophore, and the emission changed sensitively upon saccharide complexation. The binding constants for various saccharides were determined by fluorescence titration and demonstrated that the binding selectivity and affinity of the engineered Con As are comparable to those of native Con A. The red shift of the emission maximum, the decrease in the fluorescence anisotropy of the dansyl unit, and the increase in the twisted intramolecular charge transfer emission caused by sugar binding to the engineered Con Ag explicitly indicate that the microenvironment of the appended fluorephores changes from a restricted and relatively hydrophobic environment into a ranther freely mobile and hydrophilic environment.
机译：详细介绍了一种新的通用方法，即光亲和标记后修饰（PPALM），用于基于天然存在的糖结合蛋白凝集素构建荧光糖生物传感器。通过使用光亲和标记技术，将被掩蔽的反应性位点主动定向掺入凝集素中，随后进行去掩蔽，然后化学修饰以产生荧光凝集素。在这项研究中设计并合成了两种光亲和标记试剂。具有通过二硫键连接到甘露糖苷单元的光反应性位点的标记试剂与凝集素伴刀豆球蛋白A（ConA）的糖结合口袋结合。光照射后，甘露糖苷单元通过还原而裂解。由此产生的独特的硫醇基团用各种荧光基团（丹磺酰基，香豆素或二甲基氨基苯甲酸酯衍生物）进行位点特异性修饰，得到荧光的Con As。标记位点的特征是蛋白酶催化的消化，然后进行HPLC，MALDI-TOF MS和串联质谱。这些方法表明光标记步骤显着地是特异性的。在带有荧光团的工程Con A中观察到强荧光，并且在糖络合后发射灵敏地变化。通过荧光滴定法测定各种糖类的结合常数，证明工程改造的Con As的结合选择性和亲和力与天然Con A相当。发射最大值的红移，丹磺酰基的荧光各向异性降低糖与工程Con Ag的结合所引起的扭曲的分子内电荷转移发射的增加明确表明，附加的荧光团的微环境从受限制的相对疏水的环境变为自由流动和亲水的另一环境。

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