Development of an Efficient FRET-Based Ratiometric Uranium Biosensor

Abstract

The dispersion of uranium in the environment can pose a problem for the health of humans and other living organisms. It is therefore important to monitor the bioavailable and hence toxic fraction of uranium in the environment, but no efficient measurement methods exist for this. Our study aims to fill this gap by developing a genetically encoded FRET-based ratiometric uranium biosensor. This biosensor was constructed by grafting two fluorescent proteins to both ends of calmodulin, a protein that binds four calcium ions. By modifying the metal-binding sites and the fluorescent proteins, several versions of the biosensor were generated and characterized in vitro. The best combination results in a biosensor that is affine and selective for uranium compared to metals such as calcium or other environmental compounds (sodium, magnesium, chlorine). It has a good dynamic range and should be robust to environmental conditions. In addition, its detection limit is below the uranium limit concentration in drinking water defined by the World Health Organization. This genetically encoded biosensor is a promising tool to develop a uranium whole-cell biosensor. This would make it possible to monitor the bioavailable fraction of uranium in the environment, even in calcium-rich waters.

Keywords: FRET; calmodulin; genetically encoded biosensor; metal-binding; protein engineering; selectivity; sensing; sensitivity; uranium.

Figure 1

Schematic representation of the different biosensors. The donor is eCFP; CaM is the calmodulin of Arabidopsis thaliana with four metal-binding sites shown by ★ with or without mutations in one, two, three or four binding sites; L is a linker of five amino acids; M13 corresponds to the CaM-binding peptide of myosin light-chain kinase; the acceptor is eYFP or Citrine.

Figure 2

Fluorescence spectra of biosensor 1 with calcium or uranyl obtained with an excitation wavelength of 440 nm. (a) Fluorescence measured in arbitrary units (a.u.) with no metal (dashed lines) and 80 µM of calcium or 25 µM of uranyl (solid lines). (b) Fluorescence ratio obtained with fluorescence at 525 nm and at 476 nm as a function of the concentration of metal (calcium or uranyl). Measurements were performed in triplicate, and SD values are represented as error bars.

Figure 3

Fluorescence spectra of biosensor S2I with calcium (a) or uranyl (b) obtained with an excitation wavelength of 440 nm. Fluorescence measured in arbitrary units (a.u.) with no metal (dashed lines) and 80 µM of calcium or 25 µM of uranyl (solid lines).

Figure 4

Fluorescence spectra of mutated biosensors with calcium obtained with an excitation wavelength of 440 nm. Fluorescence measured in arbitrary units (a.u.) with no metal (dashed lines) and 80 µM of calcium (solid lines).

Figure 5

Fluorescence spectra of mutated biosensors with uranyl obtained with an excitation wavelength of 440 nm. (a) Fluorescence measured in arbitrary units (a.u.) with no metal (dashed lines) and 25 µM of uranyl (solid lines). (b) Fluorescence ratios obtained with fluorescence at 525 nm and at 476 nm as a function of the concentration of uranyl with biosensor 1 (●), biosensor ∆1 (●), biosensor ∆1∆2∆3 (●) or biosensor ∆1∆2∆3∆4 (●). Measurements were performed in triplicate, and SD values are represented as error bars.

Figure 6

Fluorescence ratio obtained with fluorescence at 525 nm and at 476 nm for biosensor 1 as a function of chloride concentration at pH 7.4 (a) or as a function of pH ranging from 2.5 to 11 in the presence of 135 mM of chloride (b). The excitation wavelength was 440 nm.

Figure 7

Biosensor Cit (a) and biosensor Cit ∆1∆2∆3∆4 (b) were analyzed in fluorescence with 80 µM of calcium or with 25 µM of uranyl with an excitation wavelength of 440 nm. Fluorescence measured in arbitrary units (a.u.) with no metal (dashed lines) and calcium or uranyl (solid lines).

Figure 8

Biosensor Cit ∆1∆2∆3∆4 was analyzed in fluorescence in the presence of varying concentrations of uranyl with an excitation wavelength of 440 nm. The curve represents the fluorescence ratios (F_525nm/F_476nm) as a function of uranyl concentrations. Measurements were performed in triplicate, and SD values are represented as error bars.