Construction of artificial signal transducers on a lectin surface by post-photoaffinity-labeling modification for fluorescent saccharide biosensors
Construction of artificial signal transducers on a lectin surface by post-photoaffinity-labeling modification for fluorescent saccharide biosensors
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DOI：
10.1002/chem.200304925
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发表时间：
2003-08-04
影响因子：
4.3
通讯作者：
Hamachi, I
中科院分区：
文献类型：
--
作者：
Nagase, T;Nakata, E;Hamachi, I
详细描述了一种新的通用方法，即光亲和标记后修饰（PPALM），用于构建基于天然存在的糖结合蛋白、凝集素的荧光糖生物传感器。通过使用光亲和标记技术将掩蔽的反应位点定向掺入凝集素中，然后进行去掩蔽，然后进行化学修饰以产生荧光凝集素。本研究设计并合成了两种光亲和标记试剂。具有通过二硫键连接至甘露糖苷单元的光反应位点的标记试剂与凝集素刀豆球蛋白 A (Con A) 的糖结合袋结合。光照射后，甘露糖苷单元通过还原被裂解。由此产生的独特硫醇基团被各种荧光基团（丹酰基、香豆素或二甲氨基苯甲酸酯衍生物）进行位点特异性修饰，以提供荧光Con As。通过蛋白酶催化消化、随后进行 HPLC、MALDI-TOF MS 和串联质谱分析来表征标记位点；这些方法表明光标记步骤具有显着的位点特异性。在带有荧光团的工程刀豆 A 中观察到强烈的荧光，并且发射在糖络合时发生敏感的变化。通过荧光滴定测定了各种糖的结合常数，并证明了工程Con A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、丹酰基单元荧光各向异性的降低以及糖与工程Con A结合引起的扭曲分子内电荷转移发射的增加明确表明附加荧光团的微环境从受限且相对疏水的环境转变为相当自由移动和亲水的环境环境。
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 (Con A). 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, coumarin, 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 A explicitly indicate that the microenvironment of the appended fluorophores changes from a restricted and relatively hydrophobic environment into a rather freely mobile and hydrophilic environment.