LSSmScarlet, dCyRFP2s, dCyOFP2s and CRISPRed2s, Genetically Encoded Red Fluorescent Proteins with a Large Stokes Shift

Abstract

Genetically encoded red fluorescent proteins with a large Stokes shift (LSSRFPs) can be efficiently co-excited with common green FPs both under single- and two-photon microscopy, thus enabling dual-color imaging using a single laser. Recent progress in protein development resulted in a great variety of novel LSSRFPs; however, the selection of the right LSSRFP for a given application is hampered by the lack of a side-by-side comparison of the LSSRFPs’ performance. In this study, we employed rational design and random mutagenesis to convert conventional bright RFP mScarlet into LSSRFP, called LSSmScarlet, characterized by excitation/emission maxima at 470/598 nm. In addition, we utilized the previously reported LSSRFPs mCyRFP1, CyOFP1, and mCRISPRed as templates for directed molecular evolution to develop their optimized versions, called dCyRFP2s, dCyOFP2s and CRISPRed2s. We performed a quantitative assessment of the developed LSSRFPs and their precursors in vitro on purified proteins and compared their brightness at 488 nm excitation in the mammalian cells. The monomeric LSSmScarlet protein was successfully utilized for the confocal imaging of the structural proteins in live mammalian cells and multicolor confocal imaging in conjugation with other FPs. LSSmScarlet was successfully applied for dual-color two-photon imaging in live mammalian cells. We also solved the X-ray structure of the LSSmScarlet protein at the resolution of 1.4 Å that revealed a hydrogen bond network supporting excited-state proton transfer (ESPT). Quantum mechanics/molecular mechanics molecular dynamic simulations confirmed the ESPT mechanism of a large Stokes shift. Structure-guided mutagenesis revealed the role of R198 residue in ESPT that allowed us to generate a variant with improved pH stability. Finally, we showed that LSSmScarlet protein is not appropriate for STED microscopy as a consequence of LSSRed-to-Red photoconversion with high-power 775 nm depletion light.

LSSmScarlet2 and LSSmScarlet3, Chemically Stable Genetically Encoded Red Fluorescent Proteins with a Large Stokes’ Shift

“…To develop enhanced LSSRFPs, LSSmScarlet and its pH-stable mutant LSSmScarlet/R198H were subjected to six rounds of directed molecular evolution using error-prone PCR. We chose LSSmScarlet/R198H as a template for mutagenesis due to its high pH stability with a pKa value of 2.4 [ 9 ]. During each round of evolution, we screened for mutants exhibiting the highest pH stability when expressed in E. coli cells.…”

“…The excited state proton transfer (ESPT) can explain a large Stokes’ shift in LSSmScarlet2 similar to the parental LSSmScarlet [ 9 ]. We observed a large Stokes’ shift in LSSmScarlet2, i.e., the excitation peak was shifted by 128 nm relative to the emission peak.…”

“…However, mCRISPRed is less stable at alkaline pH than at lower pH values since its fluorescence decreases at pH values higher than 7 with a pKa of 9.8. LSSmScarlet is stable at alkaline pH, but under acidic conditions, it has two pH transitions with pKa values of 1.91 and 5.78 [ 9 ]. It is known that the pH in different organelles inside mammalian cells may vary from 4.5 to 8.0 [ 10 ].…”

Dual-expression system for blue fluorescent protein optimization

“…The OSER assay is a more accessible method for characterization of monomeric state of FPs in comparison to other well-established analytical methods, such as size-exclusion chromatography 16 , 37 and analytical centrifugation 38 . However, in contrast to analytical methods, the OSER assay may not be suitable for the absolute quantification of monomeric state.…”

Tuning the Sensitivity of Genetically Encoded Fluorescent Potassium Indicators through Structure-Guided and Genome Mining Strategies

“…Proteins were expressed and purified using the pBAD/HisB arabinose-inducible system (Invitrogen) in TOP10 cells (Invitrogen) cultured in RM medium as previously described. , Before measurements, the proteins were dialyzed against various buffers depending on the subsequent measurement conditions (see sections below) and stored at 4 °C.…”