Engineering of a Brighter Variant of the FusionRed Fluorescent Protein Using Lifetime Flow Cytometry and Structure-Guided Mutations

Abstract

The development of fluorescent proteins (FPs) has revolutionized biological imaging. FusionRed, a monomeric red FP (RFP), is known for its low cytotoxicity and correct localization of target fusion proteins in mammalian cells but is limited in application by low fluorescence brightness. We report a brighter variant of FusionRed, "FR-MQV," which exhibits an extended fluorescence lifetime (2.8 ns), enhanced quantum yield (0.53), higher extinction coefficient (∼140 000 M −1 cm −1 ), increased radiative rate constant, and reduced nonradiative rate constant with respect to its precursor. The properties of FR-MQV derive from three mutationsM42Q, C159V, and the previously identified L175M. A structure-guided approach was used to identify and mutate candidate residues around the para-hydroxyphenyl and the acylimine sites of the chromophore. The C159V mutation was identified via lifetime-based flow cytometry screening of a library in which multiple residues adjacent to the para-hydroxyphenyl site of the chromophore were mutated. The M42Q mutation is located near the acylimine moiety of the chromophore and was discovered using site-directed mutagenesis guided by X-ray crystal structures. FR-MQV exhibits a 3.4-fold higher molecular brightness and a 5-fold increase in the cellular brightness in HeLa cells [based on fluorescence-activated cell sorting (FACS)] compared to FusionRed. It also retains the low cytotoxicity and high-fidelity localization of FusionRed, as demonstrated through assays in mammalian cells. These properties make FR-MQV a promising template for further engineering into a new family of RFPs.

Characterizing Dark State Kinetics and Single Molecule Fluorescence of FusionRed and FusionRed-MQ at Low Irradiances

“…28,52 FusionRed is a plausible candidate for SMLM imaging, 55 and our investigation suggests FusionRed-MQ is a better choice for such applications owing to its 1.8-fold higher quantum yield and higher kDSC. 53 The methods and results of this work can be extended to the characterization of other fluorophores with appropriate dark state kinetic models, or incorporated into multi-parametric screening technologies to select FPs with high rates of blinking for methods like SOFI.…”

“…53 This was verified by employing a high-energy 438 nm pulse (~2 s; 50% duty) with a continuous 560 nm excitation scheme which resulted in distinct reversible photoswitching for FusionRed variants with a Cys residue at position 159. 53 High energy 438 nm light prompts a return to the fluorescent state from a dark state, suggesting that the lower energy 560 nm excitation minimally perturbs the ground state recovery process. 53 Together with findings and from other studies, including crystal structure data, indicate a possible interconversion of the FusionRed chromophore from a fluorescent cis to a dark trans isomer.…”

“…Cell growth and protein purification: FusionRed and FusionRed-MQ in the pBad-His plasmid were transformed into the E. coli Top10 strain via heat shock and grown for 45-60 minutes in LB media in a shaker at 37 C and 230 rpm. 53 The transformants were plated on an agar plates with 100 g/mL ampicillin and 0.2% arabinose (Sigma Aldrich) overnight at 37 C. Colored colonies were grown in 200 mL 2XYT (VWR) liquid cultures with 100 g/mL ampicillin for 1-3 hours at 37 C and 230 rpm to an OD of 0.6.…”

“…It was demonstrated in our previous work that the D state can be depopulated efficiently using a 438 nm light. 53 The arrow labels kex, kIC, krad, kDSC and kGSR indicate the rate constants for excitation, non-radiative (internal conversion), radiative emission, S1 to dark state conversion and dark state to S0 recovery, respectively. Permanent photobleaching from the S1 and the D states are ignored in the regime of low irradiances.…”

“…28,52 Recovery from a dark to a fluorescent state is often a consequence of conformational switching such as a dark-trans to fluorescent-cis isomerization of the FP chromophore. 53 Since such conformational switches are often energetically controlled, the excitation dependence of kGSR may originate from the absorption of the excitation photons by dark state species and/or the rise of local temperature due to high irradiance. 28,52,54 FusionRed and its sibling TagRFP-T exhibit fluorescence intermittency in live-cell imaging using TIRF microscopy with camera acquisition timescales of 50 ms. 55 The study demonstrated the potential to achieve a theoretical spatial resolution beyond the diffraction limit (~25-30 nm) with FusionRed using SMLM methods like BALM and SOFI.…”