Red fluorescent proteins (RFPs) are often probes of choice for living tissue microscopy and whole-body imaging. When choosing a specific RFP variant, the priority may be given to the fluorescence brightness, maturation rate, monomericity, excitation/emission wavelengths, and low toxicity, which are rarely combined in an optimal way in a single protein. If additional requirements such as prolonged fluorescence lifetime and/or blinking ability are applied, the available repertoire of probes could dramatically narrow. Since the entire diversity of conventional single-component RFPs belongs to just a few phylogenetic lines (DsRed-, eqFP578- and eqFP611-derived being the major ones), it is not unexpected that their advantageous properties are split between close homologs. In such cases, a systematic mutagenetic analysis focusing on variant-specific amino acid residues can shed light on the origins of the distinctness between related RFPs and may aid in consolidating their strengths in new RFP variants. For instance, the protein FusionRed, despite being efficient in fluorescence labeling thanks to its good monomericity and low cytotoxicity, has undergone considerable loss in fluorescence brightness/lifetime compared to the parental mKate2. In this contribution, we describe a fast-maturing monomeric RFP designed semi-rationally based on the mKate2 and FusionRed templates that outperforms both its parents in terms of molecular brightness, has extended fluorescence lifetime, and displays a spontaneous blinking pattern that is promising for nanoscopy use.

The three dimensional structure of DiB3-F53L/F74L/L129M – a triple mutant of the fluorescent non-covalent complex of a genetically engineered variant of the bacterial protein lipocalin Blc with the synthetic GFP-like chromophore M739, has been studied by molecular dynamics methods. It was found that the chromophore binding site in the complex is similar to DiB1 and differs from the alternative site in DiB3. The complex is characterized by increased fluorescence brightness compared to those with other genetically engineered variants of lipocalin, which makes it one of the promising markers of biological objects in cell biology.

Objective: In the development of biomarker systems for cell biology, Blc lipocalin proved to be a promising scaffold for binding the fluorogen M739. This work presents the results of the molecular dynamics study of the 3D structure of the Blc lipocaline triple mutant—DiB3-F53L/F74L/L129M with a synthetic GFP-like chromophore M739. The complex is characterized by a higher fluorescence brightness compared to other complexes of genetically engineered variants of lipocalin, which makes it one of the promising biomarkers for cell research. Methods: The starting model of the protein was based on the X-ray 3D structure of DiB1 by introducing the appropriate amino acid substitutions on a stereographic station. The search for the main binding site in the triple mutant DiB3-F53L/F74L/L129M was performed by MD calculations by introducing the chromophore molecule deep into the binding cavity of the Blc protein. The MD calculations were performed in the all-atom approximation at 300 K, based on the CHARMM36 force field using an explicit solvent model. Results and Discussion: The significant brightness increase characteristic of the DiB3-F53L/F74L/L129M complex compared to DiB3 provides stronger binding of the M739 chromophore with increased solvent protection. The three-dimensional structure of the binding protein has the shape of a β-barrel, consisting of eight antiparallel β-segments with an α-helical fragment at the C-terminal region. Four loops at the N-terminus form an oval entrance (8 × 11 Å) into a narrow elongated cavity of the protein with a depth of 19 Å. The protein cavity provides specific binding of the tricyclic chromophore M739 with increased shielding from solvent. The position of the chromophore is stabilized by three H-bonds with the side chains of Asn76, Trp139 and Gln141. Conclusions: The work presents the results of the molecular dynamics study of the DiB3-F53L/F74L/L129M—Blc lipocalin triple mutant in complex with chromophore. The chromophore-binding site of the protein was found to correspond to the X-ray crystal structure of the related DiB1 complex and is different from the alternative binding site in the parent DiB3 complex.

The three-dimensional structure of the antigen-binding fragment (Fab) of the monoclonal antibody LNKB-2 in complex with the synthetic antigenic nonapeptide of human interleukin-2 (IL-2; Lys-Pro-Leu-Glu-Glu-Val-Leu-Asn-Leu-O) was determined by X-ray diffraction at a resolution of 2.6 Å in the crystal space group P212121. The peptide adopts a somewhat distorted α-helical conformation, close to that of fragment 64–72 of the IL-2 antigen. Four out of the six hypervariable loops in the antigen-binding site of the Fab fragment are involved in nonapeptide association through hydrogen bonding, salt bridge formation, and hydrophobic interactions. Moreover, Tyr residues of an antibody play an important role in antigen–antibody recognition.

The rapid development of new microscopy techniques for cell biology has exposed the need for genetically encoded fluorescent tags with special properties. Fluorescent biomarkers of the same color and spectral range and different fluorescent lifetimes (FLs) became useful for fluorescent lifetime image microscopy (FLIM). One such tag, the green fluorescent protein BrUSLEE (Bright Ultimately Short Lifetime Enhanced Emitter), having an extremely short subnanosecond component of fluorescence lifetime (FL~0.66 ns) and exceptional fluorescence brightness, was designed for FLIM experiments. Here, we present the X-ray structure and discuss the structure-functional relations of BrUSLEE. Its development from the EGFP (enhanced green fluorescent proteins) precursor (FL~2.83 ns) resulted in a change of the chromophore microenvironment due to a significant alteration in the side chain conformations. To get further insight into molecular details explaining the observed differences in the photophysical properties of these proteins, we studied their structural, dynamic, and electric properties by all-atom molecular-dynamics simulations in an aqueous solution. It has been shown that compared to BrUSLEE, the mobility of the chromophore in the EGFP is noticeably limited by nonbonded interactions (mainly H-bonds) with the neighboring residues.

The three-dimensional structure of DiB3, a fluorescent noncovalent complex of a genetically engineered variant of the bacterial protein lipocalin Blc with the synthetic GFP-like chromophore M739, was determined by molecular mechanics calculations. Compared to the crystal structure of the related DiB1 complex, an alternative binding site for the M739 chromophore was identified in the structure of the DiB3 complex.

The three-dimensional structure of DiB3, a fluorescent non-covalent complex of a genetically engineered variant of the bacterial protein lipocalin Blc with a synthetic GFP-like chromophore M739, was determined by the calculation method of molecular mechanics. Compared to the crystal structure of the related DiB1 complex, an alternative binding site for the M739 chromophore was found in the structure of the DiB3 complex.

For the whole GFP family, a few cases, when a single mutation in the chromophore environment strongly inhibits maturation, were described. Here we study EYFP-F165G - a variant of the enhanced yellow fluorescent protein - obtained by a single F165G replacement, and demonstrated multiple fluorescent states represented by the minor emission peaks in blue and yellow ranges (similar to 470 and similar to 530 nm), and the major peak at similar to 330 nm. The latter has been assigned to tryptophan fluorescence, quenched due to excitation energy transfer to the mature chromophore in the parental EYFP protein. EYFP-F165G crystal structure revealed two general independent routes of post-translational chemistry, resulting in two main states of the polypeptide chain with the intact chromophore forming triad (similar to 85%) and mature chromophore (similar to 15%). Our experiments thus highlighted important stereochemical role of the 165th position strongly affecting spectral characteristics of the protein. On the basis of the determined EYFP-F165G three-dimensional structure, new variants with similar to 2-fold improved brightness were engineered. (C) 2021 The Authors. Published by Elsevier B.V. on behalf of Research Network of Computational and Structural Biotechnology.

The crystal structure of monomeric red fluorescent protein FusionRed (λex/λem 580/608 mn) has been determined at 1.09 Å resolution and revealed two alternative routes of post-translational chemistry, resulting in distinctly different products. The refinement occupancies suggest the 60:40 ratio of the mature Met63-Tyr64-Gly65 chromophore and uncyclized chromophore-forming tripeptide with the protein backbone cleaved between Met63 and the preceding Phe62 and oxidized Cα-Cβ bond of Tyr64. We analyzed the structures of FusionRed and several related red fluorescent proteins, identified structural elements causing hydrolysis of the peptide bond, and verified their impact by single point mutagenesis. These findings advance the understanding of the post-translational chemistry of GFP-like fluorescent proteins beyond the canonical cyclization-dehydration-oxidation mechanism. They also show that impaired cyclization does not prevent chromophore-forming tripeptide from further transformations enabled by the same set of catalytic residues. Our mutagenesis efforts resulted in inhibition of the peptide backbone cleavage, and a FusionRed variant with ~30% improved effective brightness.

Super-resolution fluorescent imaging in living cells remains technically challenging, largely due to the photodecomposition of fluorescent tags. The recently suggested protein-PAINT is the only super-resolution technique available for prolonged imaging of proteins in living cells. It is realized with complexes of fluorogen-activating proteins, expressed as fusions, and solvatochromic synthetic dyes. Once photobleached, the dye in the complex is replaced with a fresh fluorogen available in the sample. With suitable kinetics, this replacement creates fluorescence blinking required for attaining super-resolution and overcomes photobleaching associated with the loss of an irreplaceable fluorophore. Here we report on the rational design of two protein-PAINT tags based on the 1.58 Å crystal structure of the DiB1:M739 complex, an improved green-emitting DiB3/F74V:M739 and a new orange-emitting DiB3/F53L:M739. They outperform previously reported DiB-based tags to become best in class biomarkers for protein-PAINT. The new tags advance protein-PAINT from the proof-of-concept to a reliable tool suitable for prolonged super-resolution imaging of intracellular proteins in fixed and living cells and two-color PAINT-like nanoscopy with a single fluorogen.

Fluorescent tags of various natures are widely used in cell biology for visualization and study processes in living organisms including gene expression, localization and migration of proteins and cells of interest, determination of the vital intracellular characteristics such as pH, ion concentration, temperature, etc. The review presents an overview of the three dimensional organization as well as advantages and disadvantages of the most promising fluorescent molecular instruments that have been widely used in biology.

Carbohydrate recognition is established as a property of lectins and implicated in many functions including immunity and defense against pathogens. Many lectins are characterized and proposed for various applications owing to the above said recognition. The crystal structure of a lectin from Pleurotus ostreatus has been determined and shown to be calcium dependent. The overall structure is a tandem repeat of two β-jelly roll domains, a new fold for lectins. The calcium dependence of sugar binding is analyzed in-detail through isothermal titration calorimetry. The serendipitous observation of malonate and glycerol, the intentional N-Acetyl-D-galactosamine, D-Galactose and L-Rhamnose binding to Pleurotus ostreatus lectin by Ca2+ coordination revealed that the binding site is promiscuous. Among these sugars, Rhamnose binding found to be thermodynamically most favourable. In all these structures, a vicinal diol motif, one at axial and the other at equatorial positions could be established as a specific requirement for binding. Interestingly, when compared with other calcium mediated lectin structures; this geometric requirement is found conserved. This observation could lead to the conclusion that lectins are not 'molecule specific' but 'geometry specific' so that any molecule not necessarily a sugar may be recognized by this lectin if the geometry exists.

GFP-like proteins from lancelets (lanFPs) is a new and least studied group that already generated several outstanding biomarkers (mNeonGreen is the brightest FP to date) and has some unique features. Here, we report the study of four homologous lanFPs with GYG and GYA chromophores. Until recently, it was accepted that the third chromophore-forming residue in GFP-like proteins should be glycine, and efforts to replace it were in vain. Now, we have the first structure of a fluorescent protein with a successfully matured chromophore that has alanine as the third chromophore-forming residue. Consideration of the protein structures revealed two alternative routes of posttranslational transformation, resulting in either chromophore maturation or hydrolysis of GYG/GYA tripeptide. Both transformations are catalyzed by the same set of catalytic residues, Arg88 and Glu35–Wat–Glu211 cluster, whereas the residues in positions 62 and 102 shift the equilibrium between chromophore maturation and hydrolysis.

The discovery of fluorescent proteins (FP) in 1962 and the following design of genetically encoded biomarkers and biosensors revolutionized the study of living systems. At present, researchers have access to FPs of a wide range of colors with a wide variety of specialized properties for visualization of biological processes in vivo using high-resolution spectroscopy techniques. GFP-like fluorescent proteins are widely used in cell biology as markers of various biological targets in a living cell. With the development of methods for visualizing processes in living organisms, the scientists have directed their efforts to designing new bright and photostable biomarkers of different colors with improved photophysical characteristics. The X-ray study plays an important role in design of the new biomarkers with improved properties. It allows us to determine the structure–function relations as a guide for directed change of the FPs properties meeting the requirements of modern research methods. The review presents the basic structures of the chromophores of the GFP-like fluorescent proteins determined by the X-ray method.

BACKGROUND The pesticidal properties of many Kunitz-type inhibitors have been reported previously; however, the mechanism of action is not well established. In this study, the activity of alocasin against Aedes aegypti is demonstrated and the structure-activity relationship of this Kunitz-type inhibitor is explained through X-ray structure analyses. RESULTS Alocasin was purified from mature rhizomes of Alocasia as a single polypeptide chain of ∼ 20 kDa. The structure at 2.5 Å resolution revealed a Kunitz-type fold, but variation in the loop regions makes this structure unique; one loop with a single disulfide bridge is replaced by a long loop with two bridges. Alignment of homologous sequences revealed that this long loop contains a conserved Arg residue and modeling studies showed interaction with the catalytic Ser residue of trypsin-like enzymes. The anti-Aedes aegypti activity of alocasin is examined and discussed in detail. The in vitro activity of alocasin against midgut proteases of Aedes aegypti showed profound inhibition. Further, morphological changes in larvae upon treatment with alocasin revealed its activity against Ae. aegypti. Docking studies of alocasin with trypsin (5G1), a midgut protease involved in the development cycle and blood meal digestion, illustrated its insecticidal activity. CONCLUSION The three-dimensional structure of alocasin was determined and its structure-function relationship established for its anti Ae. aegypti activity. © 2018 Society of Chemical Industry.

The green fluorescent protein WasCFP with a tryptophan-based chromophore (Thr65-Trp66- Gly67) exhibits considerable pH-dependent changes of spectral properties related to the reversible processes of tryptophan ionization and protonation. The crystal structure of WasCFP at pH 10.0, 8.0, and 5.5 was determined in our earlier studies. The spatial structure of WasCFP at an extremely low pH of 2.0 has been determined by X-ray diffraction with a resolution of 1.3 Å in the present study. Synchronous changes in the conformation of amino acid residue side chains in the environment of the chromophore and the related changes in the local hydrogen bond network involving the chromophore were shown to occur at sequential pH changes from 10.0 to 2.0. A quantum chemical study of the effect of interactions between the chromophore and the key amino acid residues from its immediate environment has been performed.

Brighter near-infrared (NIR) fluorescent proteins (FPs) are required for multicolor microscopy and deep-tissue imaging. Here, we present structural and biochemical analyses of three monomeric, spectrally distinct phytochrome-based NIR FPs, termed miRFPs. The miRFPs are closely related and differ by only a few amino acids, which define their molecular brightness, brightness in mammalian cells, and spectral properties. We have identified the residues responsible for the spectral red-shift, revealed a new chromophore bound simultaneously to two cysteine residues in the PAS and GAF domains in blue-shifted NIR FPs, and uncovered the importance of amino acid residues in the N-terminus of NIR FPs for their molecular and cellular brightness. The novel chromophore covalently links the N-terminus of NIR FPs with their C-terminal GAF domain, forming a topologically closed knot in the structure, and also contributes to the increased brightness. Based on our studies, we suggest a strategy to develop spectrally distinct NIR FPs with enhanced brightness.