Significant Expansion of the Fluorescent Protein Chromophore through the Genetic Incorporation of a Metal-Chelating Unnatural Amino Acid

摘要

Genetic incorporation of metal-binding unnatural amino acids (UAA) is a powerful method for protein sensor design,metalloenzyme engineering, and protein NMR spectroscopy. However, this method is currently underused by chemical biologists, because of the complex synthetic routes to UAAs. Herein, we report a one-step, high-yield enzymatic route for the synthesis of a novel UAA bearing an 8-hydroxyquinoline group (HqAla, Scheme 1), which forms highly stable complexes with most transition metal ions. By substituting the Tyr residue of the fluorophore in diverse fluorescent proteins (FPs) with HqAla, we show for the first time that UAA incorporation can result in significantly red-shifted excitation and emission spectra. We solved the crystal structure of superfolder GFP (sfGFP) with HqAla in its chromophore, revealing the formation of a novel 8-hydroxy-quinolin-imidazolinone (HQI) chromophore (Figure 2), which has a significantly larger conjugated π-system in comparison to the parental 4-(p-hydroxybenzylidene)-5-imi-dazolinone (HBI) chromophore found in Aequorea victoria green fluorescent protein (GFP). Our results indicate that HqAla incorporation into the FP fluorophore gives it unique metal-chelating and metal-ion-sensing abilities. Among all biologically relevant metal ions, only Zn~(II) ion binding to HQI causes a significant increase (7.2-fold) in fluorescence. This selective turn-on FP sensor was then applied for Zn~(II) ion sensing in vivo.