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Biphotochromic fluorescent protein SAASoti contains five cysteine residues in its sequence and a V127T point mutation transforms it to the monomeric form, mSAASoti. These cysteine residues are located far from the chromophore and might control its properties only allosterically. The influence of individual, double and triple cysteine substitutions of mSAASoti on fluorescent parameters and phototransformation reactions (irreversible green-to-red photoconversion and reversible photoswitching) is studied. A set of mSAASoti mutant forms (C21N, C117S, C71V, C105V, C175A, C21N/C71V, C21N/C175A, C21N/C71G/C175A) is obtained by site-directed mutagenesis. We demonstrate that the C21N variant exists in a monomeric form up to high concentrations, the C71V substitution accelerates photoconversion to the red form and the C105V variant has the maximum photoswitching rate. All C175A-containing variants demonstrate different photoswitching kinetics and decreased photostability during subsequent switching cycles compared with other considered systems. Classical molecular dynamic simulations reveal that the F177 side chain located in the vicinity of the chromophore is considerably more flexible in the mSAASoti compared with its C175A variant. This might be the explanation of the experimentally observed slowdown the thermal relaxation rate, i.e., trans-cis isomerization of the chromophore in mSAASoti upon C175A substitution., (© 2021. The Author(s).)

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.

Photoconvertible fluorescent proteins (PCFPs) are widely used as markers for the visualization of intracellular processes and for sub-diffraction single-molecule localization microscopy. Although wild type of a new photoconvertible fluorescent protein SAASoti tends to aggregate, we succeeded, via rational mutagenesis, to obtain variants that formed either tetramers or monomers. We compare two approaches: one is based on the structural similarity between SAASoti and Kaede, which helped us to identify a single point mutation (V127T) at the protein's hydrophobic interface that leads to monomerization. The other is based on a chemical modification of amino groups of SAASoti with succinic anhydride, which converts the protein aggregates into monomers. Mass-spectrometric analysis helped us to identify that the modification of a single ε-amino group of lysine K145 in the strongly charged interface AB was sufficient to convert the protein into its tetrameric form. Furthermore, site-directed mutagenesis was used to generate mutants that proved to be either monomeric or tetrameric, both capable of rapid green-to-red photoconversion. This allows SAASoti to be used as a photoconvertible fluorescent marker for in vivo cell studies.

Förster resonance energy transfer (FRET) sensors are widely used for the detection of protease activity in vitro and in vivo. Usually they consist of a FRET pair connected with a polypeptide linker containing a specific cleavage site for the relevant protease. Use of the fluorescent proteins as components of the FRET pair allows genetic encoding of such sensors and solves the problem of their delivery into live cells and animals. There are several ways to improve the properties of such sensors, mainly to increase FRET efficiency and therefore the dynamic range. One of the ways to achieve this is to use a non-fluorescent chromoprotein as an acceptor. Molecular dynamic simulations may assist in the construction of linker structures connecting donor and acceptor molecules. Estimation of the orientation factor κ 2 can be obtained by methods based on quantum theory and combined quantum mechanics/molecular mechanics approaches. The linker can be structured by hydrophobic interactions, bringing it into a closed conformation that shortens the distance between donor and acceptor and, consequently, increases FRET efficiency. We analyzed the effects of different linker structures on the detection of caspase-3 activity using a non-fluorescent acceptor. Also we have constructed the Tb 3+ - TagRFP sensor in which a complex of the terbium ion and terbium-binding peptide is used as a donor. This allowed us to use the unique property of lanthanide ions-fluorescence lifetime up to milliseconds-to perform measurements with time delay and exclude the nanosecond-order fluorescence. Using our systems as a starting point, by changing the recognition site in the linker it is possible to perform imaging of different protease activity in vitro or in vivo.

We characterize computationally a red fluorescent protein (RFP) with the chromophore (Chro) sandwiched between two aromatic tyrosine rings in a triple-decker motif. According to the original proposal [ J. Phys. Chem. Lett. 2013 , 4 , 1743 ], such a tyrosine-chromophore-tyrosine π-stacked construct can be accommodated in the green fluorescent protein (GFP). A recent study [ ACS Chem. Biol. 2016 , 11 , 508 ] attempted to realize the triple-decker motif and obtained an RFP variant called mRojoA-VYGV with two tyrosine residues surrounding the chromophore. The crystal structure showed that only a tyrosine-chromophore pair was involved in π-stacking, whereas the second tyrosine was oriented perpendicularly, edge-to-face with respect to the chromophore. We propose a more promising variant of this RFP with a perfect triple-decker unit achieved by introducing additional mutations in mRojoA-VYGV. The structures and optical properties of model proteins based on the structures of mCherry and mRojoA are characterized computationally by QM(DFT)/MM. The electronic transitions in the protein-bound chromophores are computed by high-level quantum chemical methods. According to our calculations, the triple-decker chromophore unit in the new RFP variant is stable within the protein and its optical bands are red-shifted with respect to the parent mCherry and mRojoA species.

We report on novel variants of the flavin-based fluorescent protein iLOV with absorption and emission optical bands shifted to the longer wavelengths relative to their precursor. First attempts (J. Phys. Chem. B 2015, 119, 5176; 2016, 120, 3344) to improve spectral properties of iLOV, a fluorescent marker in living cells, showed that single point mutation Q489K could lead to a promising variant, but its realization was not successful due to unfavorable conformation of the flexible lysine side chain pointing away from the chromophore. The results of molecular simulations presented in this work evidence that location of the charged lysine residue near the chromophore isoalloxazine ring can be fixed by introducing additional mutations in iLOV. Several suggested protein variants are characterized by using classical and QM/MM molecular dynamics simulations followed by optimization of structures in the ground and excited states. Transition energies between the S 0 and S 1 states are computed by using the advanced quantum chemical methods TD-DFT, SOS-CIS(D), and XMCQDPT2. According to our simulations, the chromophore-containing pockets for the red-shifted iLOV variants containing lysine either at position 392 (V392K/F410V/A426S) or at position 489 (Q489K/L470T) should be stable and exhibit absorption and emission bands red-shifted by 40-50 nm relative to iLOV.

This article describes the genetically encoded caspase-3 FRET-sensor based on the terbium-binding peptide, cleavable linker with caspase-3 recognition site, and red fluorescent protein TagRFP. The engineered construction performs two induction-resonance energy transfer processes: from tryptophan of the terbium-binding peptide to Tb(3+) and from sensitized Tb(3+) to acceptor--the chromophore of TagRFP. Long-lived terbium-sensitized emission (microseconds), pulse excitation source, and time-resolved detection were utilized to eliminate directly excited TagRFP fluorescence and background cellular autofluorescence, which lasts a fraction of nanosecond, and thus to improve sensitivity of analyses. Furthermore the technique facilitates selective detection of fluorescence, induced by uncleaved acceptor emission. For the first time it was shown that fluorescence resonance energy transfer between sensitized terbium and TagRFP in the engineered construction can be studied via detection of microsecond TagRFP fluorescence intensities. The lifetime and distance distribution between donor and acceptor were calculated using molecular dynamics simulation. Using this data, quantum yield of terbium ions with binding peptide was estimated.

The iLOV protein is one of the most promising members of the family of engineered flavin-based fluorescent proteins (FbFPs), considered as an alternative to the green fluorescent protein family. We modeled the spectral properties of iLOV using quantum chemistry, quantum mechanics/molecular mechanics (QM/MM), and molecular dynamics approaches. Computational results predict that the conserved Gln489 side chain in iLOV adopts two almost equally populated conformations, Glnin and Glnout, altering hydrogen bonding near the flavin chromophore. Formation of the flavinN5-Gln489 and flavinO4-Gln489 hydrogen bonds in the case of Glnin accounts for the pronounced shifts of the flavin absorption and fluorescence maxima to the longer wavelengths. Following these results, we propose to introduce a point mutation in iLOV, Q489K, with the aim to obtain a more red-shifted variant. According to our simulations, this mutation should lead to a considerable, about 50 nm, red shift of the absorption and emission band maxima, thus introducing a new color in the FbFP palette.

Chromoprotein asFP595 and its A143G variant called kindling fluorescent protein (KFP) are among the chronologically first species for which trans-cis chromophore isomerization has been proposed as a driving force of photoswitching. In spite of long-lasting efforts to characterize the route between protein conformations referring to the trans and cis forms of the chromophore, the molecular mechanism of this transformation is still under debate. We report the results of computational studies of the trans-cis isomerization of the anionic and neutral chromophore inside the protein matrices in the ground electronic state for both variants, asFP595 and KFP. Corresponding free energy profiles (potentials of mean force) were evaluated by using molecular dynamics simulations with the quantum mechanical-molecular mechanical (QM/MM) forces. The computed free energy barrier for the cis-trans ground state (thermal) isomerization reaction is about 2 kcal/mol higher in KFP than that in asFP595. These results provide interpretation of experimental studies on thermal relaxation from the light-induced activation of fluorescence of these proteins and correctly show that the A143G mutation in asFP595 noticeably increases the lifetime of the fluorescence species.