Noncovalent Association Thermodynamics of Turn-On Fluorescent Probes with Human Serum Albumin: Dual-Concentration Ratio Method

Efficient quantification of the affinity of a drug and the targeted protein is critical for strategic drug design. Among the various molecules, turn-on fluorescent probes are the most promising signal transducers to reveal the binding strength and site-specificity of designed drugs. However, the conventional method of measuring the binding ability of turn-on fluorescent probes by using the fractional occupancy under the law of mass action is time-consuming and a massive sample is required. Here, we report a new method, called dual-concentration ratio method, for quantifying the binding affinity of fluorescent probes and human serum albumin (HSA). Temperature-dependent fluorescence intensity ratios of a one-to-one complex (L ⋅ HSA) for a turn-on fluorescent probe (L), e. g., ThT (thioflavin T) or DG (dansylglycine), with HSA at two different values of [L]₀/[HSA]₀ under the constraint [HSA]₀>[L]₀ were collected. The van't Hoff analysis on these association constants further resulted in the thermodynamic properties. Since only two samples at different [L]₀/[HSA]₀ are required without the need of [L]₀/[HSA]₀ at a wide range, the dual-concentration ratio method is an easy way to greatly reduce the amounts of fluorescent probes and proteins, as well as the acquisition time.     

Engineering of a Red Fluorogenic Protein/Merocyanine Complex for Live-Cell Imaging

A reengineered human cellular retinol binding protein II (hCRBPII), a 15-kDa protein belonging to the intracellular lipid binding protein (iLBP) family, generates a highly fluorescent red pigment through the covalent linkage of a merocyanine aldehyde to an active site lysine residue. The complex exhibits “turn-on” fluorescence, due to a weakly fluorescent aldehyde that “lights up” with subsequent formation of a strongly fluorescent merocyanine dye within the binding pocket of the protein. Cellular penetration of merocyanine is rapid, and fluorophore maturation is nearly instantaneous. The hCRBPII/merocyanine complex displays high quantum yield, low cytotoxicity, specificity in labeling organelles, and compatibility in both cancer cell lines and yeast cells. The hCRBPII/merocyanine tag is brighter than most common red fluorescent proteins.     

Design,Synthesis, and Biological Evaluation of Novel Fluorescent Probes Targeting the 18-kDa Translocator Protein

A series of fluorescent probes from the 6-chloro-2-phenylimidazo[1,2-a]pyridine-3-yl acetamides ligands featuring the 7-nitro-2-oxa-1,3-diazol-4-yl (NBD) moiety has been synthesized and biologically evaluated for their fluorescence properties and for their binding affinity to the 18-kDa translocator protein (TSPO). Spectroscopic studies including UV/Vis absorption and fluorescence measurements showed that the synthesized fluorescent probes exhibit favorable spectroscopic properties, especially in nonpolar environments. In vitro fluorescence staining in brain sections from lipopolysaccharide (LPS)-injected mice revealed partial colocalization of the probes with the TSPO. The TSPO binding affinity of the probes was measured on crude mitochondrial fractions separated from rat brain homogenates in a [¹¹C]PK11195 radioligand binding assay. All the new fluorescent probes demonstrated moderate to high binding affinity to the TSPO, with affinity (K_i) values ranging from 0.58 nM to 3.28 μM. Taking these data together, we propose that the new fluorescent probes could be used to visualize the TSPO.     

Add and Go: FRET Acceptor for Live-Cell Measurements Modulated by Externally Provided Ligand

A substantial number of genetically encoded fluorescent sensors rely on the changes in FRET efficiency between fluorescent cores, measured in ratiometric mode, with acceptor photobleaching or by changes in fluorescence lifetime. We report on a modulated FRET acceptor allowing for simplified one-channel FRET measurement based on a previously reported fluorogen-activating protein, DiB1. Upon the addition of the cell-permeable chromophore, the fluorescence of the donor-fluorescent protein mNeonGreen decreases, allowing for a simplified one-channel FRET measurement. The reported chemically modulated FRET acceptor is compatible with live-cell experiments and allows for prolonged time-lapse experiments with dynamic energy transfer evaluation.     

Light‐Activated Reversible Imine Isomerization: Towards a Photochromic Protein Switch

Mutants of cellular retinoic acid‐binding protein II (CRABPII), engineered to bind all‐trans‐retinal as an iminium species, demonstrate photochromism upon irradiation with light at different wavelengths. UV light irradiation populates the cis‐imine geometry, which has a high pK_a, leading to protonation of the imine and subsequent “turn‐on” of color. Yellow light irradiation yields the trans‐imine isomer, which has a depressed pK_a, leading to loss of color because the imine is not protonated. The protein‐bound retinylidene chromophore undergoes photoinduced reversible interconversion between the colored and uncolored species, with excellent fatigue resistance.     

Chemical Synthesis of Homogeneous Glycoproteins for the Study of Glycoprotein Quality Control System

The glycoprotein quality control system exists in the endoplasmic reticulum to maintain protein homeostasis and prevent accumulation of aberrant glycoproteins. Folding sensor enzyme uridine diphosphate (UDP) glucose : glycoprotein glucosyltransferase (UGGT) plays an important role in this system through its ability to discriminate immature or misfolded glycoproteins from native ones. UGGT transfers a glucose residue to a glycoprotein containing Man₉GlcNAc₂ (M9; Man=mannose, GlcNAc=N-acetyl-D -glucosamine) N-glycan only when the glycoprotein has not attained a native form. We chemically prepared homogeneous glycoproteins containing M9 N-glycan in the native form as well as in misfolded forms and examined them as substrates of UGGT. Glucose transfer to misfolded glycoproteins was clearly observed by LC-MS, but glycoproteins in the native form were barely glucosylated. Furthermore, we constructed an in vitro glycoprotein folding system in the presence of UGGT and found out that all folding intermediates which appeared during folding were also glucosylated. Through these experiments, we demonstrated the usefulness of chemically synthesized homogeneous glycoproteins as probes to gain insights into the molecular basis of the glycoprotein quality control system.     

Mutagenic stabilization of the photocycle intermediate of green fluorescent protein (GFP)

The optical spectra of the Aequorea victoria green fluorescent protein (GFP) are governed by an equilibrium between three different chromophore states. Mutants that predominantly show either the protonated (A) or the deprotonated (B) form of the chromophore have previously been described. In contrast, the I form, which is formed by rapid excited-state deprotonation of the A form of the chromophore, has only been described as an obligatory photochemical intermediate. We report the design of a new GFP mutant with a stabilized I form. For this purpose, we introduced two isosteric point mutations, Thr203Val and Glu222Gln, that selectively raise the potential energy of both the A and the B form. Knowledge of the absorption spectrum of the I form at room temperature allows the detailed analysis of concentration dependent changes in bulk wild-type(wt)-GFP spectra, as well as the determination of the dimerization constant of GFP. This information expands the use of GFP to that of a spectral probe for protein concentration. We determined energy differences between the chromophore ground states in the monomer and the dimer and reconstructed part of the potential energy surface.     

Investigation of the Fuzzy Complex between RSV Nucleoprotein and Phosphoprotein to Optimize an Inhibition Assay by Fluorescence Polarization

The interaction between Respiratory Syncytial Virus phosphoprotein P and nucleoprotein N is essential for the formation of the holo RSV polymerase that carries out replication. In vitro screening of antivirals targeting the N-P protein interaction requires a molecular interaction model, ideally consisting of a complex between N protein and a short peptide corresponding to the C-terminal tail of the P protein. However, the flexibility of C-terminal P peptides as well as their phosphorylation status play a role in binding and may bias the outcome of an inhibition assay. We therefore investigated binding affinities and dynamics of this interaction by testing two N protein constructs and P peptides of different lengths and composition, using nuclear magnetic resonance and fluorescence polarization (FP). We show that, although the last C-terminal Phe₂₄₁ residue is the main determinant for anchoring P to N, only longer peptides afford sub-micromolar affinity, despite increasing mobility towards the N-terminus. We investigated competitive binding by peptides and small compounds, including molecules used as fluorescent labels in FP. Based on these results, we draw optimized parameters for a robust RSV N-P inhibition assay and validated this assay with the M76 molecule, which displays antiviral properties, for further screening of chemical libraries.     

Exploring the Effects of Mutagenesis on FusionRed by Using Excited-State QM/MM Dynamics and Classical Force Field Simulations**

Fluorescent proteins (FPs) are a powerful tool for examining tissues, cells, and subcellular components in vivo and in vitro. FusionRed is a particular FP variant mutated from mKate2 that, in addition to lower cytotoxicity and aggregation rates, has shown potential for acting as a tunable photoswitch. This was posited to stem partially from the presence of a bulky side chain at position 158 and a further stabilizing residue at position 157. In this work, we apply computational techniques including classical molecular dynamics (MD) and combined quantum mechanics/molecular mechanics simulations (QM/MM) to explore the effect of mutagenesis at these locations in FusionRed on the chromophore structure, the excited-state surface, and relative positional stability of the chromophore in the protein pocket. We find specific connections between the statistical sampling of the underlying protein structure and the nonradiative decay mechanisms from excited-state dynamics. A single mutation (C158I) that restricts the motion of the chromophore through a favorable hydrophobic interaction corresponds to an increase in fluorescence quantum yield (FQY), while a second rescue mutation (C158I-A157N) partially restores the flexibility of the chromophore and photoswitchability with favorable water interactions on the surface of the protein that counteracts the original interaction. We suggest that applying this understanding of structural features that inhibit or favor rotation on the excited state can be applied for rational design of new, tunable and red photoswitches.     

Destruction of shells and release of a protein from microcapsules consisting of non-biodegradable polyelectrolytes

Using fluorescent spectroscopy, we examined degradation of PAH/PSS microcapsule shells and release of a protein from them. The processes were controlled by various concentrations of NaCl and (NH₄)₂SO₄ and levels of pH: 5 and 7.4. We found that a high concentration of sodium chloride (2 М) causes essential dissociation of PAH from the upper shell layer and explained this by the layer loosening under the ionic strength. Using the optical spectroscopy, we determined amount of a microcapsule polyelectrolyte (PAH) and found that less than 20% of it can be released into 2М NaCl solution, and only 2% can be released into the water medium. Increase in solution pH up to 7.4 causes peeling of PAH too, however, temperature increase up to 37°С decreases this effect due to the structuring and compacting of the shell demonstrated by electron-microscopic studies. And finally we found that a scarce release of an encapsulated protein from the microcapsules does not depend on the presence of salts in the medium, their concentrations and a medium pH.     

Combined Mutagenesis and Kinetics Characterization of the Bilin‐Binding GAF Domain of the Protein Slr1393 from the Cyanobacterium Synechocystis PCC6803

The gene slr1393 from Synechocystis sp. PCC6803 encodes a protein composed of three GAF domains, a PAS domain, and a histidine kinase domain. GAF3 is the sole domain able to bind phycocyanobilin (PCB) as chromophore and to accomplish photochemistry: switching between a red‐absorbing parental and a green‐absorbing photoproduct state (λ_max=649 and 536 nm, respectively). Conversions in both directions were followed by time‐resolved absorption spectroscopy with the separately expressed GAF3 domain of Slr1393. Global fit analysis of the recorded absorbance changes yielded three lifetimes (3.2 μs, 390 μs, and 1.5 ms) for the red‐to‐green conversion, and 1.2 μs, 340 μs, and 1 ms for the green‐to‐red conversion. In addition to the wild‐type (WT) protein, 24 mutated proteins were studied spectroscopically. The design of these site‐directed mutations was based on sequence alignments with related proteins and by employing the crystal structure of AnPixJg2 (PDB ID: 3W2Z), a Slr1393 orthologous from Anabaena sp. PCC7120. The structure of AnPixJg2 was also used as template for model building, thus confirming the strong structural similarity between the proteins, and for identifying amino acids to target for mutagenesis. Only amino acids in close proximity to the chromophore were exchanged, as these were considered likely to have an impact on the spectral and dynamic properties. Three groups of mutants were found: some showed absorption features similar to the WT protein, a second group showed modified absorbance properties, and the third group had lost the ability to bind the chromophore. The most unexpected result was obtained for the exchange at residue 532 (N532Y). In vivo assembly yielded a red‐absorbing, WT‐like protein. Irradiation, however, not only converted it into the green‐absorbing form, but also produced a 660 nm, further‐red‐shifted absorbance band. This photoproduct was fully reversible to the parental form upon green light irradiation.     

The mRubyFT Protein,Genetically Encoded Blue-to-Red Fluorescent Timer

Genetically encoded monomeric blue-to-red fluorescent timers (mFTs) change their fluorescent color over time. mCherry-derived mFTs were used for the tracking of the protein age, visualization of the protein trafficking, and labeling of engram cells. However, the brightness of the blue and red forms of mFTs are 2–3- and 5–7-fold dimmer compared to the brightness of the enhanced green fluorescent protein (EGFP). To address this limitation, we developed a blue-to-red fluorescent timer, named mRubyFT, derived from the bright mRuby2 red fluorescent protein. The blue form of mRubyFT reached its maximum at 5.7 h and completely transformed into the red form that had a maturation half-time of 15 h. Blue and red forms of purified mRubyFT were 4.1-fold brighter and 1.3-fold dimmer than the respective forms of the mCherry-derived Fast-FT timer in vitro. When expressed in mammalian cells, both forms of mRubyFT were 1.3-fold brighter than the respective forms of Fast-FT. The violet light-induced blue-to-red photoconversion was 4.2-fold less efficient in the case of mRubyFT timer compared to the same photoconversion of the Fast-FT timer. The timer behavior of mRubyFT was confirmed in mammalian cells. The monomeric properties of mRubyFT allowed the labeling and confocal imaging of cytoskeleton proteins in live mammalian cells. The X-ray structure of the red form of mRubyFT at 1.5 Å resolution was obtained and analyzed. The role of the residues from the chromophore surrounding was studied using site-directed mutagenesis.     

Design of Lanthanide Fingers: Compact Lanthanide‐Binding Metalloproteins

Lanthanides have interesting chemical properties; these include luminescent, magnetic, and catalytic functions. Toward the development of proteins incorporating novel functions, we have designed a new lanthanide‐binding motif, lanthanide fingers. These were designed based on the Zif268 zinc finger, which exhibits a ββα structural motif. Lanthanide fingers utilize an Asp₂Glu₂ metal‐coordination environment to bind lanthanides through a tetracarboxylate peptide ligand. The iterative design of a general lanthanide‐binding peptide incorporated the following key elements: 1) residues with high α‐helix and β‐sheet propensities in the respective secondary structures; 2) an optimized big box α‐helix N‐cap; 3) a Schellman α‐helix C‐cap motif; and 4) an optional D ‐Pro‐Ser type II’ β‐turn in the β‐hairpin. The peptides were characterized for lanthanide binding by circular dichroism (CD), NMR, and fluorescence spectroscopy. In all instances, stabilization of the peptide secondary structures resulted in an increase in metal affinity. The optimized protein design was a 25‐residue peptide that was a general lanthanide‐binding motif; this binds all lanthanides examined in a competitive aqueous environment, with a dissociation constant of 9.3 μM for binding Er³⁺. CD spectra of the peptide‐lanthanide complexes are similar to those of zinc fingers and other ββα proteins. Metal binding involves residues from the N‐terminal β‐hairpin and the C terminal α‐helical segments of the peptide. NMR data indicated that metal binding induced a global change in the peptide structure. The D ‐Pro‐Ser type II’ β‐turn motif could be replaced by Thr–Ile to generate genetically encodable lanthanide fingers. Replacement of the central Phe with Trp generated genetically encodable lanthanide fingers that exhibited terbium luminescence greater than that of an EF‐hand peptide.     

Switching between Ultrafast Pathways Enables a Green-Red Emission Ratiometric Fluorescent-Protein-Based Ca2+ Biosensor

Ratiometric indicators with long emission wavelengths are highly preferred in modern bioimaging and life sciences. Herein, we elucidated the working mechanism of a standalone red fluorescent protein (FP)-based Ca²⁺ biosensor, REX-GECO1, using a series of spectroscopic and computational methods. Upon 480 nm photoexcitation, the Ca²⁺-free biosensor chromophore becomes trapped in an excited dark state. Binding with Ca²⁺ switches the route to ultrafast excited-state proton transfer through a short hydrogen bond to an adjacent Glu80 residue, which is key for the biosensor’s functionality. Inspired by the 2D-fluorescence map, REX-GECO1 for Ca²⁺ imaging in the ionomycin-treated human HeLa cells was achieved for the first time with a red/green emission ratio change (ΔR/R₀) of ~300%, outperforming many FRET- and single FP-based indicators. These spectroscopy-driven discoveries enable targeted design for the next-generation biosensors with larger dynamic range and longer emission wavelengths.     

Photosensitized Oxidative Dimerization at Tyrosine by a Water-Soluble 4-Amino-1,8-naphthalimide

The oxidation of proteins generates reactive amino acid (AA) residue intermediates, leading to protein modification and cross-linking. Aerobic studies with peptides and photosensitizers allow for the controlled generation of reactive oxygen species (ROS) and reactive AA residue intermediates, providing mechanistic insights as to how natural protein modifications form. Such studies have inspired the development of abiotic methods for protein modification and crosslinking, including applications of biomedical importance. Dityrosine linkages derived from oxidation at tyrosine (Tyr) residues represent one of the more well-understood oxidation-induced modifications. Here we demonstrate an aerobic, visible light-dependent oxidation reaction of Tyr-containing substrates promoted by a water-soluble 4-amino-1,8-naphthalimide-based photosensitizer. The developed procedure converts Tyr-containing substrates into o,o’-Tyr-Tyr linked dimers. The regioselectively formed o,o’-Tyr-Tyr linkage is consistent with dimeric standards prepared using a known enzymatic method. A crossover study with two peptides provides a statistical mixture of three distinct o,o’-Tyr-Tyr linked dimers, supporting a mechanism that involves Tyr residue oxidation followed by intermolecular combination.     

The Red‐/Green‐Switching GAF3 of Cyanobacteriochrome Slr1393 from Synechocystis sp. PCC6803 Regulates the Activity of an Adenylyl Cyclase

Cyanobacteriochromes (CBCRs) are photoreceptors in cyanobacteria that present a bilin chromophore‐binding GAF domain as a photochromic element to control the activity of a downstream enzyme or regulator. CBCR Slr1393 from Synechocystis PCC 6803 carries three GAF domains, but only the third one binds phycocyanobilin covalently. Slr1393 shows photochromicity between red and green absorbing states and regulates a C‐terminally located histidine kinase. In this work, we fused this third GAF domain to an adenylyl cyclase (AC) from Microcoleus chthonoplastes PCC7420 that in its genuine form is under blue‐light control from a LOV domain. A series of RGS‐AC variants were constructed with various lengths of the linkers between RGS and AC. Assays in vitro and in living Escherichia coli cells (AC‐deletion mutant) demonstrated that the activity of AC was light regulated, namely, the red‐light‐converted form of RGSΔ14‐Δ4AC (in vitro) was about three times more active than the green‐light‐converted form. Expression of the fusion protein RGSΔ14‐Δ4AC in vivo again showed highest light regulation with at least threefold amplification of the AC function. In some experiments, even tenfold higher activity was observed, which indicated that the protein, if expressed under in vivo conditions, was part of the E. coli physiological conditions and thereby subjected to more complex and variable regulation through other E. coli inherent factors.     

Site-specific labeling of proteins by using biotin protein ligase conjugated with fluorophores

Biotin protein ligase (BPL) mediates the covalent attachment of biotin to a specific lysine residue of biotin carboxyl carrier protein (BCCP). This biotinylation in Sulfolobus tokodaii is unique in that BPL forms a tight complex with the product, biotinylated BCCP, and this property was exploited for fluorescent labeling of a membrane protein. Thus, the truncated form of BCCP (BCCPΔ100, 69 residues) was fused to either the N or C terminus of the bradykinin B2 receptor (B2R). The resulting fusion proteins, BCCPΔ100-B2R and B2R-BCCPΔ100, respectively, were separately expressed in mammalian HEK293 cells, and labeled with BPL conjugated with a fluorophore: either fluorescein, DyLight549 or green fluorescent protein. The fusion proteins were biotinylated and bound to BPL, thereby giving rise to strong fluorescence along the periphery of the cell. Some were capable of binding bradykinin and an antagonist. When stimulated with the former, the receptor translocated to the cytosol; this suggests that the labeled receptor retains its integrity in terms of ligand-binding and translocation.     

Impact of Double Covalent Binding of BV in NIR FPs on Their Spectral and Physicochemical Properties

Understanding the photophysical properties and stability of near-infrared fluorescent proteins (NIR FPs) based on bacterial phytochromes is of great importance for the design of efficient fluorescent probes for use in cells and in vivo. Previously, the natural ligand of NIR FPs biliverdin (BV) has been revealed to be capable of covalent binding to the inherent cysteine residue in the PAS domain (Cys15), and to the cysteine residue introduced into the GAF domain (Cys256), as well as simultaneously with these two residues. Here, based on the spectroscopic analysis of several NIR FPs with both cysteine residues in PAS and GAF domains, we show that the covalent binding of BV simultaneously with two domains is the reason for the higher quantum yield of BV fluorescence in these proteins as a result of rigid fixation of the chromophore in their chromophore-binding pocket. We demonstrate that since the attachment sites are located in different regions of the polypeptide chain forming a figure-of-eight knot, their binding to BV leads to shielding of many sites of proteolytic degradation due to additional stabilization of the entire protein structure. This makes NIR FPs with both cysteine residues in PAS and GAF domains less susceptible to cleavage by intracellular proteases.     

Rational design of green fluorescent protein mutants as biosensor for bacterial endotoxin

Enhanced green fluorescent protein (EGFP) was selected as asignalling scaffold protein for design of a fluorescent biosensorfor bacterial endotoxin [or lipopolysaccharide (LPS)]. Virtualmutagenesis was utilized to model EGFP variants containing bindingsites for LPS and lipid A (LA), the bioactive component of LPS.Cationic amphipathic sequences of five alternating basic andhydrophobic residues were introduced to ß-sheets locatedon the surface of EGFP barrel, in the vicinity of the chromophore.Computational methods were employed to predict binding affinityof Escherichia coli LA, to the models of virtual EGFP mutants.DNA mutant constructs of five predicted best binding EGFP variantswere expressed in COS-1 cells. The EGFP-mutant proteins exhibiteddifferential expression and variable degrees of fluorescenceyield at 508 nm. The EGFP mutants showed a range of LA bindingaffinities that corresponded to the computational predictions.LPS/LA binding to the mutants caused concentration-dependentfluorescence quenching. The EGFP mutant, G10 bearing LPS/LAamphipathic binding motif in the vicinity of the chromophore(YLSTQ_200–204