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.

Objective: Cell biology continuously shows the need for new fluorescent tags with advanced properties. The object of our current study is a new genetically encoded monomeric red fluorescent biomarker mKate2-K67R/R197H (λex/λem 579/603 mn), designed from commercial biomarker mKate2 by two R197H/K67R mutations. The mKate2 precursor, a far-red fluorescent protein, is nearly 3-fold brighter than the previously designed mKate. Compared with commercial mKate2, the double mutant mKate2-K67R/R197H (alternative names FusionRed2 and Diogenes) exhibits an additional 1.6-fold increase in fluorescence brightness and represents the next generation of extra-bright red fluorescent probes offering novel possibilities for fluorescent imaging of proteins in living cells and animals. Methods: The paper presents the results of X-ray and molecular dynamics study of new bright biomarker mKate2-K67R/R197H. Results and Discussion: The three dimensional structure of new advanced red fluorescent biomarker mKate2-K67R/R197H has been studied by X-ray method at 1.5Å resolution supported by molecular dynamics (MD) study The principal structural fold of the protein is an 11-stranded β-barrel. The nearest chromophore environment (≤ 4 Å) comprises 18 tightly packed residues. Conclusions: The MD study showed that the brightness of mKate2-K67R/R197H and its mKate2 precursor correlates with the dipole moments of the amino acid environments of the chromophores. The higher the dipole moment, the higher the brightness of biomarker.

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 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.

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.

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.

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.

WasCFP, a pH-dependent green fluorescent protein with a tryptophan-based chromophore (Thr65-Trp66-Gly67) in anionic state, was designed from a cyan precursor mCerulean. In this study, the three-dimensional structure of WasCFP has been determined by an X-ray method at pH 5.5, pH 8.0 and pH 10.0, with a resolution of 1.14, 1.25 and 1.5 Å, respectively. We show that changes in the acidity of the media are accompanied by a synchronous change of the side chain conformations of the residues in the near-chromophore environment. Subsequent changes in the local H-bond network interacting with the chromophore lead to considerable alterations in the protein spectral properties as a consequence of reversible processes of ionization-protonation of the Trp chromophore. These experimental results have been supported by quantum chemistry calculations.

The spatial structure of dimeric green fluorescent protein EGFP-K162Q with MDELYK (EGFPv) C-terminal deletion has been assigned in the P61 space group with resolution 1.34 Å by X-ray diffraction analysis. The results have been compared with X-ray diffraction data of monomeric EGFP (green biomarker with enhanced photophysical properties) assigned in another crystal space group, P212121, with resolution 1.50 and 1.35 Å. Subunits in the EGFPv dimeric structure are located at 75° angle with the contact area ∼800 Å2. The dimeric framework is stabilized by the six hydrogen bonds and central hydrophobic core of six residues. The root-mean-square deviation value for Cα atoms in 3–230 residues of the P61 and P212121 crystal structures is 0.55 Å. The differential characteristics of EGFPv-P61 structure, compared to that of P212121, is a noticeably different orientation of the Glu222 side chain, and a new conformation of the 155–159 loop fragment, characterized by deviations among the Cα atoms of superimposed structures reaching 4.6 Å for Lys156 and 5.5 Å for Lys158.