Fluorescent noncanonical amino acids (fNCAAs) could serve as starting points for the rational design of protein-based fluorescent sensors of biological activity. However, efforts toward this goal are likely hampered by a lack of atomic-level characterization of fNCAAs within proteins. Here, we describe the spectroscopic and structural characterization of five streptavidin mutants that contain the fNCAA l-(7-hydroxycoumarin-4-yl)ethylglycine (7-HCAA) at sites proximal to the binding site of its substrate, biotin. Many of the mutants exhibited altered fluorescence spectra in response to biotin binding, which included both increases and decreases in fluorescence intensity as well as red- or blue-shifted emission maxima. Structural data were also obtained for three of the five mutants. The crystal structures shed light on interactions between 7-HCAA and functional groups, contributed either by the protein or by the substrate, that may be responsible for the observed changes in the 7-HCAA spectra. These data could be used in future studies aimed at the rational design of fluorescent, protein-based sensors of small molecule binding or dissociation.

中文翻译：

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含有荧光非经典氨基酸的蛋白质中配体结合后光谱性质改变的结构起源

荧光非经典氨基酸 (fNCAAs) 可以作为合理设计基于蛋白质的生物活性荧光传感器的起点。然而，实现这一目标的努力可能会因缺乏对蛋白质内 fNCAA 的原子级表征而受到阻碍。在这里，我们描述了五种含有 fNCAA l的链霉亲和素突变体的光谱和结构特征。-(7-hydroxycoumarin-4-yl)ethylglycine (7-HCAA) 在靠近其底物生物素结合位点的位点。许多突变体响应于生物素结合而表现出改变的荧光光谱，其中包括荧光强度的增加和减少以及红移或蓝移发射最大值。还获得了五个突变体中的三个的结构数据。晶体结构揭示了 7-HCAA 和官能团之间的相互作用，由蛋白质或底物贡献，这可能是观察到的 7-HCAA 光谱变化的原因。这些数据可用于未来的研究，旨在合理设计基于蛋白质的荧光小分子结合或解离传感器。