Search

In vivoimaging of model organisms is heavily reliant on fluorescent proteins with high intracellular brightness. Here we describe a practical method for rapid optimization of fluorescent proteins via directed molecular evolution in cultured mammalian cells. Using this method, we were able to perform screening of large gene libraries containing up to 2·107independent random genes of fluorescent proteins expressed in HEK cells completing one iteration directed evolution in a course of ∼8 days. We employed this approach to develop a set of green and near-infrared fluorescent proteins with enhanced intracellular brightness. The developed near-infrared fluorescent proteins demonstrated high performance for fluorescent labeling of neurons in culture andin vivoin model organisms such asC.elegans,Drosophila, zebrafish, and mice. Spectral properties of the optimized near-infrared fluorescent proteins enabled crosstalk-free multicolor imaging in combination with common green and red fluorescent proteins, as well as dual-color near-infrared fluorescence imaging. The described method has a great potential to be adopted by protein engineers due to its simplicity and practicality. We also believe that the new enhanced fluorescent proteins will find wide application forin vivomulticolor imaging of small model organisms.

Pyridoxal 5′-phosphate (PLP)-dependent transaminases catalyze the stereospecific amino-group transfer from an amino acid or amine to ketone or keto acid. Transaminases are involved in amino acid metabolism in all organisms. Enzymes of this superfamily are widely used to develop biocatalysts for the stereoselective amination of organic compounds for fine organic synthesis. The brief biochemical characterization of thermostable fold type I PLP-dependent transaminase from the thermophilic archaeon Methanocaldococcus jannaschii DSM 2661 is reported. The crystal structure of this enzyme was determined at 1.8 A resolution. The structure of the functional dimer of the enzyme and the organization of its active site are compared with those of the close homologs.

The effect of replacements of amino-acid residues that form the interface between monomers in a dimer of the HU protein from Spiroplasma Melliferum (HUSpm) on the stability and conformational dynamics of the molecule is investigated. The behavior of single and multiple HUSpm mutants is studied by molecular dynamics. Simulation is carried out using the GROMACS software package, and OPLS is used as a force field. For each mutant, a full-atom simulation is performed with a duration of 50 ns. The data obtained show that disruption of the interface between monomers in the alpha-helical domain increase the mobility of the molecule in the DNA-binding domain.

The search of a putative physiological electron acceptor for thiocyanate dehydrogenase (TcDH) newly discovered in the thiocyanate-oxidizing bacteria

The results of the structural studies of bacterial oligopeptidase B (OpB) belonging to the least well-studied prolyl oligopeptidase family are described. The screening of crystallization conditions for mutants of the enzyme, its complexes with peptides, which mimic substrates and catalytic reaction products, and a complex with a transition state analog as an inhibitor was performed in order to determine the three-dimensional structures of OpB from Serratia proteamaculans (PSP) acting at different steps of the catalytic cycle. Crystals suitable for X-ray diffraction were grown. The X-ray diffraction data sets were collected, processed, and subjected to preliminary analysis. These X-ray diffraction data sets are suitable for obtaining the structural data necessary for the description of the catalytic cycle of bacterial ОpBs.

Histone-like HU proteins are global regulators of the topology of the bacterial genome and are involved in transcriptional regulation. These proteins are essential for the survival of the simplest free-living microorganisms of the Mollicutes class and a number of pathogenic bacteria, including Mycobacterium tuberculosis and Streptococcus pneumonia, and are promising targets for the design of new antibiotics. However, the competitive inhibition of the DNA-binding ability of HU proteins is inefficient because of high flexibility and the large size of the DNA-binding site. Hence, an alternative approach was employed based on searching for molecules targeting the interface between two monomers. The three-dimensional structure of the HU protein from Spiroplasma melliferum (HUSpm) was used as a target for the virtual screening of the compound library and for the modeling of HUSpm in complex with the inhibitor. An analysis of the molecular dynamics trajectory calculated for the HUSpm–inhibitor complex confirmed the fact that the binding of the inhibitor at the interface between monomers leads to the disruption of conformational dynamics in the region of the DNA-binding domain. The gel retardation assay showed that the inhibitor actually disturbs DNA-binding activity of the protein, thereby indicating that this is a promising approach.

The crystal structure of monomeric red fluorescent protein FusionRed (λex/λem 580/608 mn) has been determined at 1.09 A 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.

Oligopeptidases B (OpdBs) are two-domain serine peptidases with trypsin-like substrate specificity belonging to the prolyl oligopeptidase family. These enzymes are involved in the pathogenesis of trypanosomiasis, leishmaniasis, other parasitic infections, and in some cases bacterial infections and are potential targets for the development of therapeutic anti-infective agents. Only two three-dimensional structures of enzymes of this class were determined. Both these enzymes were from protozoa, whereas the structures of bacterial OpdBs are unknown. Differential scanning fluorimetry (thermofluor) was used to increase the efficiency of screening of crystallization conditions of OpdB from the bacterium Serratia proteamaculans (PSP). It was found that low-molecular-weight polyamines can stabilize PSP in solution. A crystal of PSP, which was grown by the vapor-diffusion method in the presence of spermine, was used to collect the X-ray diffraction data set to 1.88 A resolution at the synchrotron radiation source in the National Research Centre “Kurchatov Institute.”

International journal of molecular sciences 23(17), 9969 (2022). doi:10.3390/ijms23179969, The search of a putative physiological electron acceptor for thiocyanate dehydrogenase (TcDH) newly discovered in the thiocyanate-oxidizing bacteria Thioalkalivibrio paradoxus revealed an unusually large, single-heme cytochrome c (CytC552), which was co-purified with TcDH from the periplasm. Recombinant CytC552, produced in Escherichia coli as a mature protein without a signal peptide, has spectral properties similar to the endogenous protein and serves as an in vitro electron acceptor in the TcDH-catalyzed reaction. The CytC552 structure determined by NMR spectroscopy reveals significant differences compared to those of the typical class I bacterial cytochromes c: a high solvent accessible surface area for the heme group and so-called “intrinsically disordered” nature of the histidine-rich N- and C-terminal regions. Comparison of the signal splitting in the heteronuclear NMR spectra of oxidized, reduced, and TcDH-bound CytC552 reveals the heme axial methionine fluxionality. The TcDH binding site on the CytC552 surface was mapped using NMR chemical shift perturbations. Putative TcDH-CytC552 complexes were reconstructed by the information-driven docking approach and used for the analysis of effective electron transfer pathways. The best pathway includes the electron hopping through His528 and Tyr164 of TcDH, and His83 of CytC552 to the heme group in accordance with pH-dependence of TcDH activity with CytC552., Published by Molecular Diversity Preservation International, Basel

Human InsR, IGF1R, and IRR receptor tyrosine kinases (RTK) of the insulin receptor subfamily play an important role in signaling pathways for a wide range of physiological processes and are directly associated with many pathologies, including neurodegenerative diseases. The disulfide-linked dimeric structure of these receptors is unique among RTKs. Sharing high sequence and structure homology, the receptors differ dramatically in their localization, expression, and functions. In this work, using high-resolution NMR spectroscopy supported by atomistic computer modeling, conformational variability of the transmembrane domains and their interactions with surrounding lipids were found to differ significantly between representatives of the subfamily. Therefore, we suggest that the heterogeneous and highly dynamic membrane environment should be taken into account in the observed diversity of the structural/dynamic organization and mechanisms of activation of InsR, IGF1R, and IRR receptors. This membrane-mediated control of receptor signaling offers an attractive prospect for the development of new targeted therapies for diseases associated with dysfunction of insulin subfamily receptors.

The crystal structure of bacterial oligopeptidase B from Serratia proteamaculans (SpOpB) in complex with a chloromethyl ketone inhibitor was determined at 2.2 Å resolution. SpOpB was crystallized in a closed (catalytically active) conformation. A single inhibitor molecule bound simultaneously to the catalytic residues S532 and H652 mimicked a tetrahedral intermediate of the catalytic reaction. A comparative analysis of the obtained structure and the structure of OpB from Trypanosoma brucei (TbOpB) in a closed conformation showed that in both enzymes, the stabilization of the D-loop (carrying the catalytic D) in a position favorable for the formation of a tetrahedral complex occurs due to interaction with the neighboring loop from the β-propeller. However, the modes of interdomain interactions were significantly different for bacterial and protozoan OpBs. Instead of a salt bridge (as in TbOpB), in SpOpB, a pair of polar residues following the catalytic D617 and a pair of neighboring arginine residues from the β-propeller domain formed complementary oppositely charged surfaces. Bioinformatics analysis and structural modeling show that all bacterial OpBs can be divided into two large groups according to these two modes of D-loop stabilization in closed conformations.

Intrinsically disordered myelin basic protein (MBP) is one of the key autoantigens in autoimmune neurodegeneration and multiple sclerosis particularly. MBP is highly positively charged and lacks distinct structure in solution and therefore its intracellular partners are still mostly enigmatic. Here we used combination of formaldehyde-induced cross-linking followed by immunoprecipitation and liquid chromatography-tandem mass spectrometry (LC-MS/MS) to elucidate the interaction network of MBP in mammalian cells and provide the list of potential MBP interacting proteins. Our data suggest that the largest group of MBP-interacting proteins belongs to cellular proteins involved in the protein translation machinery, as well as in the spatial and temporal regulation of translation. MBP interacts with core ribosomal proteins, RNA helicase Ddx28 and RNA-binding proteins STAU1, TDP-43, ADAR-1 and hnRNP A0, which are involved in various stages of RNA biogenesis and processing, including specific maintaining MBP-coding mRNA. Among MBP partners we identified CTNND1, which has previously been shown to be necessary for myelinating Schwann cells for cell-cell interactions and the formation of a normal myelin sheath. MBP binds proteins MAGEB2/D2 associated with neurotrophin receptor p75NTR, involved in pathways that promote neuronal survival and neuronal death. Finally, we observed that MBP interacts with RNF40–a component of heterotetrameric Rnf40/Rnf20 E3 ligase complex, recruited by Egr2, which is the central transcriptional regulator of peripheral myelination. Concluding, our data suggest that MBP may be more actively involved in myelination not only as a main building block but also as a self-regulating element.

Simple Summary Oligopeptidase B is a two-domain, trypsin-like peptidase from parasitic protozoa and bacteria which belongs to the least studied group of prolyloligopeptidases. In this study, we describe for the first time a crystal structure of bacterial oligopeptidase B and compare it with those of protozoan oligopeptidases B and related prolyloligopeptidases. The enzyme was crystallized in the presence of spermine and contained a modified sequence of the interdomain linker. Both factors were key for crystallization. The structure showed an uncommon intermediate conformation with a domain arrangement intermediate between open and closed conformations found in the crystals of ligand-free and inhibitor-bound prolyloligopeptidases, respectively. To evaluate the impact of the modification and spermine in the obtained conformation, small-angle X-ray scattering was applied, which showed that in solution wild-type enzymes adopt the open conformation and spermine causes a transition to the intermediate state, while the modification is associated with a partial transition. We suggest that spermine-dependent conformational transition replicates the behavior of the enzyme in bacterial cells and the intermediate state, which is rarely detected in vitro, and might be widely distributed in vivo, and so should be considered during computational studies, including those aimed wanting to develop the small molecule inhibitors targeting prolyloligopeptidases. Abstract Oligopeptidase B (OpB) is a two-domain, trypsin-like serine peptidase belonging to the S9 prolyloligopeptidase (POP) family. Two domains are linked by a hinge region that participates in the transition of the enzyme between two major states—closed and open—in which domains and residues of the catalytic triad are located close to each other and separated, respectively. In this study, we described, for the first time, a structure of OpB from bacteria obtained for an enzyme from Serratia proteomaculans with a modified hinge region (PSPmod). PSPmod was crystallized in a conformation characterized by a disruption of the catalytic triad together with a domain arrangement intermediate between open and closed states found in crystals of ligand-free and inhibitor-bound POP, respectively. Two additional derivatives of PSPmod were crystallized in the same conformation. Neither wild-type PSP nor its corresponding mutated variants were susceptible to crystallization, indicating that the hinge region modification was key in the crystallization process. The second key factor was suggested to be polyamine spermine since all crystals were grown in its presence. The influences of the hinge region modification and spermine on the conformational state of PSP in solution were evaluated by small-angle X-ray scattering. SAXS showed that, in solution, wild-type PSP adopted the open state, spermine caused the conformational transition to the intermediate state, and spermine-free PSPmod contained molecules in the open and intermediate conformations in dynamic equilibrium.

Among industrially important pyridoxal-5’-phosphate (PLP)-dependent transaminases of fold type IV D-amino acid transaminases are the least studied. However, the development of cascade enzymatic processes, including the synthesis of D-amino acids, renewed interest in their study. Here, we describe the identification, biochemical and structural characterization of a new D-amino acid transaminase from Haliscomenobacter hydrossis (Halhy). The new enzyme is strictly specific towards D-amino acids and their keto analogs, it demonstrates one of the highest rates of transamination between D-glutamate and pyruvate. We obtained the crystal structure of the Halhy in the holo form with the protonated Schiff base formed by the K143 and the PLP. Structural analysis revealed a novel set of the active site residues that differ from the key residues forming the active sites of the previously studied D-amino acids transaminases. The active site of Halhy includes three arginine residues, one of which is unique among studied transaminases. We identified critical residues for the Halhy catalytic activity and suggested functions of the arginine residues based on the comparative structural analysis, mutagenesis, and molecular modeling simulations. We suggested a strong positive charge in the O-pocket and the unshaped P-pocket as a structural code for the D-amino acid specificity among transaminases of PLP fold type IV. Characteristics of Halhy complement our knowledge of the structural basis of substrate specificity of D-amino acid transaminases and the sequence-structure-function relationships in these enzymes.

In vivo imaging of model organisms is heavily reliant on fluorescent proteins with high intracellular brightness. Here we describe a practical method for rapid optimization of fluorescent proteins via directed molecular evolution in cultured mammalian cells. Using this method, we were able to perform screening of large gene libraries containing up to 2·107 independent random genes of fluorescent proteins expressed in HEK cells completing one iteration directed evolution in a course of ~ 8 days. We employed this approach to develop a set of green and near-infrared fluorescent proteins with enhanced intracellular brightness. The developed near-infrared fluorescent proteins demonstrated high performance for fluorescent labeling of neurons in culture and in vivo in model organisms such as C.elegans, Drosophila, zebrafish, and mice. Spectral properties of the optimized near-infrared fluorescent proteins enabled crosstalk-free multicolor imaging in combination with common green and red fluorescent proteins, as well as dual-color near-infrared fluorescence imaging. The described method has a great potential to be adopted by protein engineers due to its simplicity and practicality. We also believe that the new enhanced fluorescent proteins will find wide application for in vivo multi-color imaging of small model organisms.

Histone-like protein HU is a dimeric nucleoid-associated protein (NAP). HU is the most conserved NAP. It binds nonspecifically to duplex DNA with a preference for targeting nicked and bent DNA. HU limits the architecture of the bacterial nucleoid and its deletion is lethal for Bacillus subtilis and Mycoplasma genitalium which do not contain other NAPs. E. coli lacking HU is viable but has numerous growth defects. The effects of the HU protein on gene expression is known from microarray analysis and HU regulons were identified. In HU-deficient E. coli, absence of this DNA architectural protein causes a disorder in gene regulation; on the other hand, E. coli growth under standard conditions is almost unaltered in the absence of HU. To understand how the bacterium confronts the chromosomal disorder, we performed proteome analysis to compare protein abundances in cells containing the HU protein or not. Comparison of the proteomic profile of wild-type and HU-deficient E. coli shows how the altered gene expression influences the protein content. We show that proteome profile changes are very similar to the gene expression profile changes in HU-deficient E. coli. Several exceptions show that proteome studies are very important.

The behavior of the peptide substrates GlyArgArgGly and GlyLysArgGly, which contain arginine or lysine at the Р2 position, in the active site of oligopeptidase В from the gamma-proteobacterium Serratia proteamaculans was studied by molecular dynamics simulations. The nature of the residue at the Р2 position was shown not only to influence the position of this residue and its contacts with the amino-acid environment in the substrate-binding pocket but also to affect the conformational dynamics of the entire peptide. Only the residue at the Р1 position forms persistent contacts with the S1-substrate-binding site of the enzyme, whereas contacts of the residue at the Р2 position may vary depending on its nature. Molecular dynamics simulations are shown to complement and improve the knowledge extracted from X-ray diffraction studies that enable the determination of the three-dimensional structures of molecules in one of numerous alternative conformations.

A mutant of bacterial carboxypeptidase T from Thermoactinomyces vulgaris (CPT5) with amino-acid substitutions in the S1' specificity pocket of the active site for the residues corresponding to the S1' region of pancreatic carboxypeptidase B (Gly215Ser, Ala251Gly, Thr257Ala, Asp260Gly, Thr262Asp) was crystallized in complex with N-BOC-L-Leu. The latter occupies the S1 subsite of the active site and is a reaction product analog. The X-ray diffraction data set was collected from a crystal of CPT5 at the SPring 8 synchrotron facility. The three-dimensional structure of CPT5 was determined at 1.40 A resolution and refined to Rfact = 13.6%, Rfree = 12.5%. The binding of N-BOC-L-Leu to the S1 subsite of CPT5 leads to conformational changes in the protein concerning primarily the Сα atoms of five residues of the flexible loop at the interface between the S1 and S1' subsites and the side chains of the residues Tyr255 and Leu254 involved in the induced fit. Besides, conformational changes are observed in the active-site residues Glu277 and Asp262 and the side chains of residues located on the surface of the protein molecule. The observed changes differ from the conformational changes, which occur upon binding of N-BOC-L-Leu to wild-type CPT. This fact is indicative of the mutual influence of the S1 and S1' subsites in metallocarboxypeptidase T.

The pre-crystallization solution of the transaminase from Thermobaculum terrenum (TaTT) has been studied by small-angle X-ray scattering (SAXS). Regular changes in the oligomeric composition of the...

DNA-Binding HU proteins are critical to the maintenance of the nucleoid structure and are promising pharmacological targets for the design of new antibiotics. The virtual screening for low-molecular-weight compounds capable of specifically interacting with the dimerization signals of two mycoplasma HU proteins was performed. The stability of target protein–ligand complexes was evaluated by molecular dynamics simulation. An analysis of the amino-acid environment of the ligands shows that inhibitors targeting the conserved amino-acid sequence, the so-called dimerization signal, which is involved in the formation of HU dimers and the maintenance of their stability and is located in an interdimer beta-sheet, have the second binding site in the alpha-helical domain. The binding determinants of inhibitors at the dimer interfaces of the proteins are identical and are independent of the amino-acid sequence. Consequently, HU proteins have two sites for the formation of the dimer interface, which should be taken into account in the design of inhibitors of dimerization.

Pyridoxal-5′-phosphate (PLP)-dependent transaminases are industrially important enzymes catalyzing the stereoselective amination of ketones and keto acids. Transaminases of PLP fold type IV are characterized by (R)- or (S)-stereoselective transfer of amino groups, depending on the substrate profile of the enzyme. PLP fold type IV transaminases include branched-chain amino acid transaminases (BCATs), D-amino acid transaminases and (R)-amine:pyruvate transaminases. Recently, transaminases with a mixed type of activity were identified and characterized. Here, we report biochemical and structural characterization of a transaminase from myxobacterium Haliangium ochraceum (Hoch3033), which is active towards keto analogs of branched-chain amino acids (specific substrates for BCATs) and (R)-(+)-α-methylbenzylamine (specific substrate for (R)-amine:pyruvate transaminases). The enzyme is characterized by an alkaline pH optimum (pH 10.0–10.5) and a tolerance to high salt concentrations (up to 2 M NaCl). The structure of Hoch3033 was determined at 2.35 A resolution. The overall fold of the enzyme was similar to those of known enzymes of PLP fold type IV. The mixed type of activity of Hoch3033 was implemented within the BCAT-like active site. However, in the active site of Hoch3033, we observed substitutions of specificity-determining residues that are important for substrate binding in canonical BCATs. We suggest that these changes result in the loss of activity towards α-ketoglutarate and increase the affinity towards (R)-(+)-α-methylbenzylamine. These results complement our knowledge of the catalytic diversity of transaminases and indicate the need for further research to understand the structural basis of substrate specificity in these enzymes.

The high catalytic efficiency of enzymes under reaction conditions is one of the main goals in biocatalysis. Despite the dramatic progress in the development of more efficient biocatalysts by protein design, the search for natural enzymes with useful properties remains a promising strategy. The pyridoxal 5'-phosphate (PLP)-dependent transaminases represent a group of industrially important enzymes due to their ability to stereoselectively transfer amino groups between diverse substrates; however, the complex mechanism of substrate recognition and conversion makes the design of transaminases a challenging task. Here we report a detailed structural and kinetic study of thermostable transaminase from the bacterium Thermobaculum terrenum (TaTT) using the methods of enzyme kinetics, X-ray crystallography and molecular modeling. TaTT can convert L-branched-chain and L-aromatic amino acids as well as (R)-(+)-1-phenylethylamine at a high rate and with high enantioselectivity. The structures of TaTT in complex with the cofactor pyridoxal 5'-phosphate covalently bound to enzyme and in complex with its reduced form, pyridoxamine 5'-phosphate, were determined at resolutions of 2.19 Å and 1.5 Å, and deposited in the Protein Data Bank as entries 6GKR and 6Q8E, respectively. TaTT is a fold type IV PLP-dependent enzyme. In terms of structural similarity, the enzyme is close to known branched-chain amino acid aminotransferases, but differences in characteristic sequence motifs in the active site were observed in TaTT compared to canonical branched-chain amino acid aminotransferases, which can explain the improved binding of aromatic amino acids and (R)-(+)-1-phenylethylamine. This study has shown for the first time that high substrate specificity towards both various l-amino acids and (R)-primary amines can be implemented within one pyridoxal 5'-phosphate-dependent active site of fold type IV. These results complement our knowledge of the catalytic diversity of transaminases and indicate the need for further biochemical and bioinformatic studies to understand the sequence-structure-function relationship in these enzymes.

Creating biocatalysts for (R)-selective amination effectively is highly desirable in organic synthesis. Despite noticeable progress in the engineering of (R)-amine activity in pyridoxal-5’-phosphate-dependent transaminases of fold type IV, the specialization of the activity is still an intuitive task, as there is poor understanding of sequence-structure-function relationships. In this study, we analyzed this relationship in transaminase from Thermobaculum terrenum, distinguished by expanded substrate specificity and activity in reactions with L-amino acids and (R)-(+)-1-phenylethylamine using α-ketoglutarate and pyruvate as amino acceptors. We performed site-directed mutagenesis to create a panel of the enzyme variants, which differ in the active site residues from the parent enzyme to a putative transaminase specific to (R)-primary amines. The variants were examined in the overall transamination reactions and half-reaction with (R)-(+)-1-phenylethylamine. A structural analysis of the most prominent variants revealed a spatial reorganization in the active sites, which caused changes in activity. Although the specialization to (R)-amine transaminase was not implemented, we succeeded in understanding the role of the particular active site residues in expanding substrate specificity of the enzyme. We showed that the specificity for (R)-(+)-1-phenylethylamine in transaminase from T. terrenum arises without sacrificing the specificity for L-amino acids and α-ketoglutarate and in consensus with it.

Here we report bisphenol derivatives of fluorene (BDFs) as a new type of chemical probes targeting a histone-like HU protein, a global regulator of bacterial nucleoids, via its dimerization interface perturbation. BDFs were identified by virtual screening and molecular docking that targeted the core of DNA-binding β-saddle-like domain of the HU protein from Spiroplasma melliferum. However, NMR spectroscopy, complemented with molecular dynamics and site-directed mutagenesis, indicated that the actual site of the inhibitors’ intervention consists of residues from the α-helical domain of one monomer and the side portion of the DNA-binding domain of another monomer. BDFs inhibited DNA-binding properties of HU proteins from mycoplasmas S. melliferum, Mycoplasma gallicepticum and Escherichia coli with half-maximum inhibitory concentrations in the range between 5 and 10 µM. In addition, BDFs demonstrated antimicrobial activity against mycoplasma species, but not against E. coli, which is consistent with the compensatory role of other nucleoid-associated proteins in the higher bacteria. Further evaluation of antimicrobial effects of BDFs against various bacteria and viruses will reveal their pharmacological potential, and the allosteric inhibition mode reported here, which avoids direct competition for the binding site with DNA, should be considered in the development of small molecule inhibitors of nucleoid-associated proteins as well as other types of DNA-binding multimeric proteins.

Genetically encoded calcium indicators (GECIs) have become a widespread tool for the visualization of neuronal activity. As compared to popular GCaMP GECIs, the FGCaMP indicator benefits from calmodulin and M13-peptide from the fungi Aspergillus niger and Aspergillus fumigatus, which prevent its interaction with the intracellular environment. However, FGCaMP exhibits a two-phase fluorescence behavior with the variation of calcium ion concentration, has moderate sensitivity in neurons (as compared to the GCaMP6s indicator), and has not been fully characterized in vitro and in vivo. To address these limitations, we developed an enhanced version of FGCaMP, called FGCaMP7. FGCaMP7 preserves the ratiometric phenotype of FGCaMP, with a 3.1-fold larger ratiometric dynamic range in vitro. FGCaMP7 demonstrates 2.7- and 8.7-fold greater photostability compared to mEGFP and mTagBFP2 fluorescent proteins in vitro, respectively. The ratiometric response of FGCaMP7 is 1.6- and 1.4-fold higher, compared to the intensiometric response of GCaMP6s, in non-stimulated and stimulated neuronal cultures, respectively. We reveal the inertness of FGCaMP7 to the intracellular environment of HeLa cells using its truncated version with a deleted M13-like peptide, in contrast to the similarly truncated variant of GCaMP6s. We characterize the crystal structure of the parental FGCaMP indicator. Finally, we test the in vivo performance of FGCaMP7 in mouse brain using a two-photon microscope and an NVista miniscope, and in zebrafish using two-color ratiometric confocal imaging.

Biocatalytic copper centers are generally involved in the activation and reduction of dioxygen, with only few exceptions known. Here we report the discovery and characterization of a previously undescribed copper center that forms the active site of a copper-containing enzyme thiocyanate dehydrogenase (suggested EC 1.8.2.7) that was purified from the haloalkaliphilic sulfur-oxidizing bacterium of the genus Thioalkalivibrio ubiquitous in saline alkaline soda lakes. The copper cluster is formed by three copper ions located at the corners of a near-isosceles triangle and facilitates a direct thiocyanate conversion into cyanate, elemental sulfur, and two reducing equivalents without involvement of molecular oxygen. A molecular mechanism of catalysis is suggested based on high-resolution three-dimensional structures, electron paramagnetic resonance (EPR) spectroscopy, quantum mechanics/molecular mechanics (QM/MM) simulations, kinetic studies, and the results of site-directed mutagenesis.

It is generally accepted that the primary specificity of metallocarboxypeptidases is mainly determined by the structure of the so-called primary specificity pocket. However, the G215S/A251G/T257A/D260G/T262D mutant of carboxypeptidase T from Thermoactinomyces vulgaris (CPT) with the primary specificity pocket fully reproducing the one in pancreatic carboxypeptidase B (CPB) retained the broad, mainly hydrophobic substrate specificity of the wild-type enzyme. In order to elucidate factors affecting substrate specificity of metallocarboxypeptidases and the reasons for the discrepancy with the established views, we have solved the structure of the complex of the CPT G215S/A251G/T257A/D260G/T262D mutant with the transition state analogue N-sulfamoyl-L-phenylalanine at a resolution of 1.35 Å and compared it with the structure of similar complex formed by CPB. The comparative study revealed a previously underestimated structural determinant of the substrate specificity of metallocarboxypeptidases and showed that even if substitution of five amino acid residues in the primary specificity pocket results in its almost complete structural correspondence to the analogous pocket in CPB, this does not lead to fundamental changes in the substrate specificity of the mutant enzyme due to the differences in the structure of the mobile loop located at the active site entrance that affects the substrate-induced conformational rearrangements of the active site.

Substrate and reaction promiscuity is a remarkable property of some enzymes and facilitates the adaptation to new metabolic demands in the evolutionary process. Substrate promiscuity is also a basis for protein engineering for biocatalysis. However, molecular principles of enzyme promiscuity are not well understood. Even for the widely studied PLP-dependent transaminases of class III, the reliable prediction of the biocatalytically important amine transaminase activity is still difficult if the desired activity is unrelated to the natural activity. Here, we show that 7,8-diaminopelargonic acid transaminase (synthase), previously considered to be highly specific, is able to convert (S)-(-)-1-phenylethylamine and a number of aldehydes and diketones. We were able to characterize the (S)-amine transaminase activity of 7,8-diaminopelargonic acid transaminase from Psychrobacter cryohalolentis (Pcryo361) and analyzed the three-dimensional structure of the enzyme. New substrate specificity for α-diketones was observed, though only a weak activity towards pyruvate was found. We examined the organization of the active site and binding modes of S-adenosyl-L-methionine and (S)-(-)-1-phenylethylamine using X-ray analysis and molecular docking. We suggest that the Pcryo361 affinity towards (S)-(-)-1-phenylethylamine arises from the recognition of the hydrophobic parts of the specific substrates, S-adenosyl-L-methionine and 7-keto-8-aminopelargonic acid, and from the flexibility of the active site. Our results support the observation that the conversion of amines is a promiscuous activity of many transaminases of class III and is independent from their natural function. The analysis of amine transaminase activity from among various transaminases will help to make the sequence-function prediction for biocatalysis more reliable.

The L232H mutant of the enhanced green fluorescent protein (EGFP) was expressed and crystallized. An X-ray diffraction data set was collected from the crystals to 1.53 A resolution. An analysis of the three-dimensional structure revealed a stacking interaction between the amino-acid residues Н78 and Н232, which contributes to the fastening of the C-terminal region of the protein in the vicinity of the chromophore and influences chromophore maturation of hybrid fluorescent proteins produced by fusion of the target proteins with the C-terminus of EGFP. This hypothesis was experimentally confirmed by investigating chromophore maturation of the hybrid proteins fused to the N- and C-termini of EGFP and EGFP-L232H.

Metallocarboxypeptidases (MCPs) have been in a focus of biochemical studies since 1954 when carboxypeptidase A (CPA) was discovered as the second, after carbonic anhydrase, zinc-dependent enzyme up...

DNA-Binding HU proteins are essential for the maintenance of genomic DNA supercoiling and compaction in prokaryotic cells and are promising pharmacological targets for the design of new antibacterial agents. The virtual screening for low-molecular-weight compounds capable of specifically interacting with the DNA-recognition loop of the HU protein from the mycoplasma Spiroplasma melliferum was performed. The ability of the initially selected ligands to form stable complexes with the protein target was assessed by molecular dynamics simulation. One compound, which forms an unstable complex, was eliminated by means of a combination of computational methods, resulting in a decrease in the number of compounds that will pass to the experimental test phase. This approach can be used to solve a wide range of problems related to the search for and validation of low-molecular-weight inhibitors specific for a particular protein target.

The pre-crystallization solution of the transaminase from

Oligopeptidases B (OpdBs) are trypsin-like peptidases from protozoa and bacteria that belong to the prolyl oligopeptidase (POP) family. All POPs consist of C-terminal catalytic domain and N-terminal β-propeller domain and exist in two major conformations: closed (active), where the domains and residues of the catalytic triad are positioned close to each other, and open (non-active), where two domains and residues of the catalytic triad are separated. The interdomain interface, particularly, one of its salt bridges (SB1), plays a role in the transition between these two conformations. However, due to double amino acid substitution (E/R and R/Q), this functionally important SB1 is absent in γ-proteobacterial OpdBs including peptidase from Serratia proteamaculans (PSP). In this study, molecular dynamics was used to analyze inter- and intradomain interactions stabilizing PSP in the closed conformation, in which catalytic H652 is located close to other residues of the catalytic triad. The 3D models of either wild-type PSP or of mutant PSPs carrying activating mutations E125A and D649A in complexes with peptide-substrates were subjected to the analysis. The mechanism that regulates transition of H652 from active to non-active conformation upon domain separation in PSP and other γ-proteobacterial OpdB was proposed. The complex network of polar interactions within H652-loop/C-terminal α-helix and between these areas and β-propeller domain, established in silico, was in a good agreement with both previously published results on the effects of single-residue mutations and new data on the effects of the activating mutations on each other and on the low active mutant PSP-K655A. Communicated by Ramaswamy H. Sarma

Pigment Epithelium-Derived Factor (PEDF) is a secreted glycoprotein belonging to the family of non-inhibitory serpins. It is known, that in cases of complicated myopia, the content of PEDF in aqueous humor of the anterior chamber is significantly reduced. Here we examined a bulk of Tenon's capsule samples obtained from various groups of myopes, to examine PEDF processing in progressive myopia. We have analyzed the distribution of full length PEDF50 and its truncated form PEDF45 in the soluble and insoluble fractions extracted from Tenon's capsule of myopic and control (non-myopic) patients using SDS-polyacrylamide gel electrophoresis, as well as monitored the proteolytic degradation of PEDF ex vivo by enzyme-linked immunosorbent assay. These results were complemented by PEDF mRNA analysis in correspondent tissues by using qPCR and immunohistochemistry analysis of PEDF distribution in normal and myopic specimens. We found that in the Tenon's capsule of patients suffering from a high myopia the level of "soluble" 45 kDa PEDF reduced by 2-fold, while the content of "insoluble" 50 kDa form of PEDF was increased by 4-fold compared to controls. Excessive amount of PEDF50 in myopic specimens have been shown to correlate with the abrogated PEDF processing rather than with an increase of its expression. Moreover, immunohistochemical staining of the myopic Tenon's capsule tissue sections revealed the halo of deposited PEDF50 in the fibroblast extracellular space. These findings suggest that in myopia limited proteolysis of PEDF is altered or abrogated. Accumulation of full-length PEDF insoluble aggregates in the fibroblast intercellular space may affect cell survival and consequently causes the destructive changes in the extracellular matrix of the eye connective tissues. As a result, the abrogation of full-length PEDF normal processing can be an important mechanism leading to biomechanical destabilization of the scleral capsule and myopia progression.