Flavin-based fluorescent proteins (FbFPs) belong to the blue-light photoreceptor protein family containing light-oxygen-voltage (LOV) sensing domains. Oxygen-independent fluorescent FbFPs outperform the well-established green fluorescent protein (GFP) family in aspects of smaller size, stability over a wider pH range, and rapid and oxygen-independent fluorophore incorporation. A FbFP derived from <i>Chlamydomonas reinhardtii </i>(CreiLOV) exhibits a high quantum yield and great photophysical properties, making it a promising platform for new fluorescent probes. Our previous study showed that CreiLOV was selectively quenched by Cu(II) ions and a binding site was proposed. Here, we probe the hypothesized copper-binding site via protein engineering. Among several mutations, the response to Zn(II) of one variant was altered. Excess Zn(II) quenched the fluorescence intensity by 95% and reduced fluorescence lifetime from 4.5 ns to 2.7 ns. Moreover, the CreiLOV mutant binds Zn(II) with a nanomolar affinity. Additionally, the affinity toward Cu(II) of the mutant is ~25 fM, which is nearly four orders of magnitude higher than that for wild type CreiLOV. Finally, we show the mutated CreiLOV is suitable as an intensity- and fluorescence lifetime-based metal sensor in live bacterial cells by demonstrating that the Zn(II)-induced fluorescence quenching is reversible over several cycles in cell suspensions and that reversible zinc-induced quenching can be detected by fluorescence microscopy.

Flavin-binding fluorescent proteins as platforms for designing new metal ion sensors

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ACS Fall 2022

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.