Abstract Body:

Flavin-based fluorescent proteins (FbFPs) are a class of small fluorescent reporters which can function without the presence of oxygen by binding locally available flavin as a chromophore,¹ a distinctive contrast to the Green Fluorescent Protein (GFP) and its relatives which require oxygen to develop their active chromophore.² However, few biosensors have been made using FbFPs compared to the broad arsenal of sensors developed using GFP. Among factors precluding extensive creation of FbFP-based sensors are the lack of highly fluorescent circular permutants of FbFP and poor understanding of the behavior of domains fused to FbFPs. Closure of these gaps in knowledge will facilitate engineering of more FbFP-based sensors by mirroring designs that use GFP and circularly permuted GFP.³

As such, we present a circular permutant of iLOV, a frequently used FbFP,⁴ which we have termed cpiLOV. Linker engineering and reintroduction of the Jα helix, a feature present in optogenetically-active LOV proteins but absent in iLOV, are necessary for fluorescence of cpiLOV. Independently, cpiLOV shows much diminished fluorescence in cells, quantum yield, protein holofraction, and thermal stability as compared to iLOV. However, we have rescued of these properties through the fusion of self-dimerizing coiled coils (leucine zippers) to the termini of cpiLOV. Interestingly, addition of leucine zippers to non-permuted iLOV in a similar fashion strongly improves each of these metrics. Each such variant retains the characteristic spectral shapes of FbFPs, with an excitation peak at ~ 450 nm and an emission peak at ~ 495nm with a shoulder at ~ 530 nm. We speculate that these dimerizing domains reduce the entropy of FbFPs’ termini, preventing the protein from visiting states in which its flavin chromophore is solvent exposed and thus less fluorescent.

To demonstrate the utility of cpiLOV, we have constructed an adaptable platform for detection of protease activity in mammalian cells. By inserting a tobacco etch virus protease (TEVp) cleavage site into the linker region of “zipped” cpiLOV we conferred protease sensitivity, in which the protein is highly fluorescent until cleaved by TEVp, upon which its fluorescence is diminished by 73 ± 10%. We show that this platform can be adapted simply to a new protease target, SARS-CoV-2 main protease (Mpro), solely by substitution of the appropriate protease cleavage site, yielding a 61 ± 17% Mpro-induced fluorescence decrease.

Circular permutation of FbFPs can enable construction of a broad palette of biosensors useful to study anaerobic systems in their native contexts. Further, understanding the impact of terminally-fused domains on the fluorescence of FbFPs bolsters further engineering efforts on these reporters.

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