Institut für Chemie, Carl von Ossietzky-Universität, Postfach 2503, 26111 Oldenburg , Germany.
Chembiochem. 2012 May 7;13(7):993-8. doi: 10.1002/cbic.201200027. Epub 2012 Apr 13.
Diaminoterephthalates with a maleimide moiety were synthesized and used as fluorescence dyes for sensing thiols. Whereas these "NiWa Blue" dyes showed no emission, the conjugate addition of a thiol to the maleimide group turned on a fluorescence at about 400 nm when irradiating the dye at 338 nm. The neuronal-calcium sensor protein recoverin possesses a single cysteine residue at position 39, which reacts with NiWa Blue, and is therefore labeled by a fluorophore with an emission at about 440 nm. In the absence of Ca(2+), irradiation at 280 nm of a tryptophan residue in close proximity to Cys-bound NiWa Blue lead to strong FRET, which was detected by emission of the dye at 440 nm. In the presence of Ca(2+), the protein holds a conformation with distal Trp and Cys residues, thus FRET of irradiated Trp to Cys-bound NiWa Blue was significantly weakened.
合成了带有马来酰亚胺部分的二氨基对苯二甲酸盐，并将其用作检测巯基的荧光染料。这些“NiWa Blue”染料没有发射，但是当用 338nm 光照射时，硫醇与马来酰亚胺基团的共轭加成会在大约 400nm 处打开荧光。神经元钙传感器蛋白 recoverin 在位置 39 处具有一个单一的半胱氨酸残基，它与 NiWa Blue 反应，因此被发射约 440nm 的荧光团标记。在没有 Ca(2+)的情况下，靠近与 Cys 结合的 NiWa Blue 的色氨酸残基在 280nm 光的照射下会导致强烈的 FRET，这可以通过染料在 440nm 处的发射来检测。在 Ca(2+)存在下，该蛋白具有一个具有远端 Trp 和 Cys 残基的构象，因此，照射的 Trp 与 Cys 结合的 NiWa Blue 的 FRET 显著减弱。
