Genetically encoded green calcium indicators (GECIs) are broadly used for visualizing calcium transients in living cells. Among the diverse family of green GECIs, the Troponin C-based family offers potential advantages, including reduced calcium buffering, smaller molecular size, linear calcium response, and low cytotoxicity. However, the Troponin C-based GECIs with inverted calcium response are less developed compared to other popular GECIs, including GCaMPs and GECOs, and, as a consequence, have several drawbacks related to low dynamic range, brightness, photostability, and calcium ion sensitivity. To address these limitations, we developed a novel GECI, called icBTnC2, which incorporates Troponin C as a sensing moiety and the new bright photostable green FP mBaoJin as a reporting moiety. icBTnC2 demonstrated an inverted fluorescent response to calcium ion binding with a Kd of 62 nM. In terms of fluorescence contrast and calcium ion affinity in vitro, icBTnC2 was comparable to the best widely used calmodulin-based GECIs from the GCaMP family. icBTnC2 demonstrated superior photostability under wide-field fluorescence microscopy exhibiting 5.5-, 4.8-, 3.2-, 2.9-, and 1.3-fold higher photobleaching half-time compared to iYTnC2, mEGFP, NCaMP7, jGCaMP8f, and mBaoJin, respectively. The icBTnC2 indicator was benchmarked against other GECIs, such as jGCaMP8f, NCaMP7, iYTnC2, and R-GECO1, for visualization of calcium transients in mammalian cells and primary neuron cultures, and tested for calcium-dependent changes in fluorescence lifetime. Finally, we solved the crystal structure of the icBTnC2 indicator at 1.55 Å resolution in the calcium-bound state and, using directed mutagenesis, proposed the molecular basis of its fluorescent response to calcium ion binding.

Research objective. To develop refolding conditions for recombinant proteins prone to aggregation and inclusion body formation during expression in Escherichia coli, using two homologs of the CRISPR effector Cas12m as an example. Materials and methods. The Cas12m genes were amplified and cloned into expression vectors with an N-terminal His-tag. The resulting plasmids were transformed into E. coli for protein production. Protein expression was induced by IPTG, after which the proteins accumulated in inclusion bodies were solubilized in a chaotropic agent and purified by immobilized metal affinity chromatography (IMAC) on an Ni-TED column with simultaneous on-column refolding using the “dual-gradient” method. Results. A comprehensive screening of expression conditions was conducted; however, in all tested bacterial strains, the proteins formed inclusion bodies. Optimization of induction conditions and the use of various fusion tags did not yield significant improvements. To address the issue of low solubility, a refolding protocol was developed, which involved immobilizing the denatured protein on Ni-TED resin. This protocol proved to be the most effective compared to traditional methods. A key aspect was the application of the “dual-gradient” system—a simultaneous decrease in the denaturant concentration and an increase in the detergent concentration. As a result, the target proteins were successfully obtained in a soluble form. Despite protein loss due to partial aggregation and the presence of contaminant proteins typical for metal-chelate chromatography, the resulting preparations were of sufficient purity for subsequent studies of functional activity. Conclusions. The proposed method outperforms traditional refolding techniques, such as dialysis or dilution, in terms of reproducibility and the yield of soluble protein. This approach can be extended to a wide range of aggregation-prone proteins and is considered a viable strategy for obtaining difficult-to-solubilize proteins in a soluble form.

This study presents the design, synthesis, and evaluation of a novel series of covalent broad-spectrum inhibitors targeting the coronavirus main protease (3CLpro). The designed compounds feature a tetrahydroquinoline (THQ) scaffold functionalized with a chloroacetamide warhead. The most potent of this series in the primary screening assay, 4bf and 5bf, exhibited low micromolar IC₅₀ values against 3CLpro of SARS-CoV-2, SARS-CoV, and MERS-CoV, thereby demonstrating significant cross-reactivity. Structural analysis via X-ray crystallography confirmed covalent binding to the catalytic Cys145 residue. Complementary molecular dynamics simulations revealed stable binding modes and key interactions, highlighting differences in flexibility and residue contacts between the top inhibitors. While in vitro cytotoxicity was observed in Vero E6 cells, acute toxicity studies in mice revealed an LD₅₀ exceeding 1000 mg/kg for the lead compounds, indicating a promising in vivo safety profile. These findings establish substituted tetrahydroquinolines as a viable scaffold for the development of broad-spectrum anticoronaviral agents.

The interaction of the ectodomain of the human coronavirus spike protein with the phospholipid monolayers formed on the aqueous subphase surface has been investigated. The changes in the molecular organization of monolayers of two neutral phospholipids—dipalmitoylphosphatidylcholine and dipalmitoylphosphatidylethanolamine—after the injection of a protein solution underneath the monolayer have been analyzed. Experiments were performed with a recombinant ectodomain of the S-protein, expressed in a CHO-K1 cell strain. Electron microscopy data showed that the protein is trimerized. Grazing incidence diffraction measurements were performed to study the influence of the trimer ectodomain of the S-protein on the structure of the dipalmitoylphosphatidylcholine monolayer. It is found that protein injection under the monolayer does not induce disturbance of the monolayer crystal structure. The experimental results obtained in X-ray studies and compression isotherm measurements indicate that the interaction with the S-protein does not result in destabilization of the monolayer for both phospholipids.

A new derivative of 4-methoxy-1,8-naphthalimide containing a chelating motif of lysine-derived mono-N-nitrilotriacetic acid effectively coordinates with Ni2+in a HEPES-buffer solution resulting in the formation of 1 : 1 ligand-metal complex. This demonstrates the possibility to stain His-labeled proteins on a gel after electrophoresis to make the analysis and purification of proteins more convenient.

The PSmOrange and PSmOrange2 fluorescent proteins undergo irreversible photoconversion from the orange to far-red form under blue light, which makes them probes of choice for protein tracking and single-molecule super-resolution imaging. However, both proteins exhibit noticeable photoconversion under 550-570 nm light used for excitation of their orange form, which complicates applications of these photoconvertible FPs in cell imaging experiments. Here, we report the next-generation PSmOrange variant, called PSmOrange3, which is characterized by minimal photoconversion under 550-570 nm light and high photoconversion contrast. PSmOrange3 undergoes efficient photoconversion from the orange (Ex/Em at 550 nm/564 nm) to far-red form (Ex/Em at 614 nm/655 nm) with 430-470 nm violet-blue light of moderate power density (3-180 mW/mm2) in a native cellular environment. The molecular brightness of orange and far-red forms of PSmOrange3 was 1.2- and 1.4-fold brighter than that of PSmOrange2. In addition, PSmOrange3 had a substantially higher photostability of the orange form but a little less photostability of the far-red form. We solved the crystal structure of PSmOrange3 at a 2.8 Å resolution, which confirmed its monomeric state and revealed the role of the introduced mutations in the properties of PSmOrange3. Using mass spectrometry we revealed the chemical structure of the PSmOrange3 chromophore before and after photoconversion. PSmOrange3 was properly localized with different protein fusions and photoconverted from the orange to far-red state inside live and fixed mammalian cells without exogenously supplied oxidants. Among all proteins of the PSmOrange series, both forms of PSmOrange3 were the brightest in the reducing environment of the mitochondrial lumen. PSmOrange3 photoconverted efficiently with blue light and almost did not photoconvert with green light, which allows investigators to excite its orange form and photoconvert it to the far-red form with different light. We demonstrated the applicability of PSmOrange3 for photoactivated localization microscopy (PALM) of tubulin microtubules using 488-nm photoconversion, achieving mean localization precision per single-molecule event of 24.6 and 23.3 nm in fixed and live mammalian cells, respectively. We believe that PSmOrange3 can represent a suitable alternative to the PSmOrange and PSmOrange2 proteins and will be a valuable addition to the repertoire of available photoconvertible fluorescent proteins.

Branched-chain amino acids (BCAAs) play an important role in the functioning of mammalian cells and the central nervous system. However, available genetically encoded indicators for BCAAs are based on Forster resonance energy transfer and have a limited dynamic range. We developed a single fluorescent protein-based sensor for BCAAs, called NeIle, which is composed of circularly permutated mNeonGreen protein inserted into the leucine-isoleucine-valine binding protein (LIVBP) from Escherichia coli bacteria. In solution, the NeIle indicator displayed a positive fluorescence response to adding isoleucine, leucine, and valin amino acids with high Delta F/F dynamic ranges of 27-, 19-, and 11-fold and the corresponding affinity values of 5.0, 2.9, and 75 mM, respectively. The spectral and biochemical properties of the NeIle indicator were characterized in solution. We characterized the brightness of the NeIle indicator in living mammalian cells, including cultured neurons. Using the NeIle indicator, we successfully visualized the dynamics of isoleucine transients in different organelles of mammalian cells. We obtained and analyzed the X-ray crystal structure of the NeIle indicator in an isoleucine-bound state. Structure-guided directed mutagenesis of the NeIle indicator revealed the basis of its fluorescence response and selectivity to isoleucine.

Human rhinovirus picornain 3C is a high-value commercial cysteine protease, which is widely used to remove affinity tags and fusion proteins during the purification of the target proteins. A variant of rhinovirus A28 picornain 3C produced in this study is not annotated in the NCBI databases, shares 79% sequence identity in the PDB, and was not previously used in the protein engineering. A protocol was developed for the isolation and purification of the protein to use it in structural studies. The initial crystallization conditions were found. The determination and analysis of the structure of rhinovirus A28 picornain 3C will provide new possibilities for performing basic research on the evolution of proteolytic enzymes and for the design of the optimal variant of this protease.

High brightness and photostability of StayGold make it a particularly attractive probe for long-term live cell imaging. However, its dimeric nature precludes its application as a fluorescent tag for some proteins. Here, we report the development and X-ray structures of a monomeric variant of StayGold (mBaoJin), which preserves the beneficial properties of its precursor while serving as a tag for structural proteins and membranes. We compare mBaoJin to other state-of-art GFPs and utilize it for super-resolution long-term live cell imaging and expansion microscopy.

Genetically encoded calcium indicators based on truncated troponin C are attractive probes for calcium imaging due to their relatively small molecular size and twofold reduced calcium ion buffering. However, the best-suited members of this family, YTnC and cNTnC, suffer from low molecular brightness, limited dynamic range, and/or poor sensitivity to calcium transients in neurons. To overcome these limitations, we developed an enhanced version of YTnC, named YTnC2. Compared with YTnC, YTnC2 had 5.7-fold higher molecular brightness and 6.4-fold increased dynamic range in vitro. YTnC2 was successfully used to reveal calcium transients in the cytosol and in the lumen of mitochondria of both mammalian cells and cultured neurons. Finally, we obtained and analyzed the crystal structure of the fluorescent domain of the YTnC2 mutant.

The mRubyFT is a monomeric genetically encoded fluorescent timer based on the mRuby2 fluorescent protein, which is characterized by the complete maturation of the blue form with the subsequent conversion to the red one. It has higher brightness in mammalian cells and higher photostability compared with other fluorescent timers. A high-resolution structure is a known characteristic of the mRubyFT with the red form chromophore, but structural details of its blue form remain obscure. In order to obtain insight into this, we obtained an S148I variant of the mRubyFT (mRubyFTS148I) with the blocked over time blue form of the chromophore. X-ray data at a 1.8 Å resolution allowed us to propose a chromophore conformation and its interactions with the neighboring residues. The imidazolidinone moiety of the chromophore is completely matured, being a conjugated π-system. The methine bridge is not oxidized in the blue form bringing flexibility to the phenolic moiety that manifests itself in poor electron density. Integration of these data with the results of molecular dynamic simulation disclosed that the OH group of the phenolic moiety forms a hydrogen bond with the side chain of the T163 residue. A detailed comparison of mRubyFTS148I with other available structures of the blue form of fluorescent proteins, Blue102 and mTagBFP, revealed a number of characteristic differences. Molecular dynamic simulations with the combined quantum mechanic/molecular mechanic potentials demonstrated that the blue form exists in two protonation states, anion and zwitterion, both sharing enolate tautomeric forms of the C=C–O− fragment. These two forms have similar excitation energies, as evaluated by calculations. Finally, excited state molecular dynamic simulations showed that excitation of the chromophore in both protonation states leads to the same anionic fluorescent state. The data obtained shed light on the structural features and spectral properties of the blue form of the mRubyFT timer.

True genetically encoded monomeric fluorescent timers (tFTs) change their fluorescent color as a result of the complete transition of the blue form into the red form over time. Tandem FTs (tdFTs) change their color as a consequence of the fast and slow independent maturation of two forms with different colors. However, tFTs are limited to derivatives of the mCherry and mRuby red fluorescent proteins and have low brightness and photostability. The number of tdFTs is also limited, and there are no blue-to-red or green-to-far-red tdFTs. tFTs and tdFTs have not previously been directly compared. Here, we engineered novel blue-to-red tFTs, called TagFT and mTagFT, which were derived from the TagRFP protein. The main spectral and timing characteristics of the TagFT and mTagFT timers were determined in vitro. The brightnesses and photoconversions of the TagFT and mTagFT tFTs were characterized in live mammalian cells. The engineered split version of the TagFT timer matured in mammalian cells at 37 °C and allowed the detection of interactions between two proteins. The TagFT timer under the control of the minimal arc promoter, successfully visualized immediate-early gene induction in neuronal cultures. We also developed and optimized green-to-far-red and blue-to-red tdFTs, named mNeptusFT and mTsFT, which were based on mNeptune-sfGFP and mTagBFP2-mScarlet fusion proteins, respectively. We developed the FucciFT2 system based on the TagFT-hCdt1-100/mNeptusFT2-hGeminin combination, which could visualize the transitions between the G1 and S/G2/M phases of the cell cycle with better resolution than the conventional Fucci system because of the fluorescent color changes of the timers over time in different phases of the cell cycle. Finally, we determined the X-ray crystal structure of the mTagFT timer and analyzed it using directed mutagenesis.