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[摘要]：The fluorescence of maleimide-functionalized diaminoterephthalate derivatives (NiWa Orange) is turned on by the conjugate addition of thiols. Three new representatives of this class of dyes with emission at 560 nm were synthesized from succinyl succinates. The mechanism of turning on the fluorescence was investigated by computational studies. Radiationless transition to an energetically low-lying excited state by a nonadiabatic interaction was the mechanism leading to absence of fluorescence prior to the reaction of the probe with its molecular target. This concept was proven by labeling cysteine-containing peptides and proteins. For example, the single cysteine residue at position 39 of the neuronal calcium sensor protein recoverin binds to NiWa Orange and turns on its fluorescence. The Ca2(+)-dependent Forster resonance energy transfer (FRET) process from a tryptophan residue in proximity to Cys39-bound NiWa Orange was used for the determination of differences in Ca2(+)-binding affinities of myristoylated and nonmyristoylated recoverin. |
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