Dmitry Dolgikh | Lomonosov Moscow State University (original) (raw)
Papers by Dmitry Dolgikh
International Journal of Molecular Sciences
Peptide pore blockers and their fluorescent derivatives are useful molecular probes to study the ... more Peptide pore blockers and their fluorescent derivatives are useful molecular probes to study the structure and functions of the voltage-gated potassium Kv1.3 channel, which is considered as a pharmacological target in the treatment of autoimmune and neurological disorders. We present Kv1.3 fluorescent ligand, GFP–MgTx, constructed on the basis of green fluorescent protein (GFP) and margatoxin (MgTx), the peptide, which is widely used in physiological studies of Kv1.3. Expression of the fluorescent ligand in E. coli cells resulted in correctly folded and functionally active GFP–MgTx with a yield of 30 mg per 1 L of culture. Complex of GFP–MgTx with the Kv1.3 binding site is reported to have the dissociation constant of 11 ± 2 nM. GFP–MgTx as a component of an analytical system based on the hybrid KcsA–Kv1.3 channel is shown to be applicable to recognize Kv1.3 pore blockers of peptide origin and to evaluate their affinities to Kv1.3. GFP–MgTx can be used in screening and pre-selection...
Biomolecules
A key event in the cytochrome c-dependent apoptotic pathway is the permeabilization of the outer ... more A key event in the cytochrome c-dependent apoptotic pathway is the permeabilization of the outer mitochondrial membrane, resulting in the release of various apoptogenic factors, including cytochrome c, into the cytosol. It is believed that the permeabilization of the outer mitochondrial membrane can be induced by the peroxidase activity of cytochrome c in a complex with cardiolipin. Using a number of mutant variants of cytochrome c, we showed that both substitutions of Lys residues from the universal binding site for oppositely charged Glu residues and mutations leading to a decrease in the conformational mobility of the red Ω-loop in almost all cases did not affect the ability of cytochrome c to bind to cardiolipin. At the same time, the peroxidase activity of all mutant variants in a complex with cardiolipin was three to five times higher than that of the wild type. A pronounced increase in the ability to permeabilize the lipid membrane in the presence of hydrogen peroxide, as mea...
Molecules
Quite a long time ago, Oleg B. Ptitsyn put forward a hypothesis about the possible functional sig... more Quite a long time ago, Oleg B. Ptitsyn put forward a hypothesis about the possible functional significance of the molten globule (MG) state for the functioning of proteins. MG is an intermediate between the unfolded and the native state of a protein. Its experimental detection and investigation in a cell are extremely difficult. In the last decades, intensive studies have demonstrated that the MG-like state of some globular proteins arises from either their modifications or interactions with protein partners or other cell components. This review summarizes such reports. In many cases, MG was evidenced to be functionally important. Thus, the MG state is quite common for functional cellular proteins. This supports Ptitsyn’s hypothesis that some globular proteins may switch between two active states, rigid (N) and soft (MG), to work in solution or interact with partners.
Biomolecules
This Special Issue of Biomolecules demonstrates the almost unlimited possibilities of modern prot... more This Special Issue of Biomolecules demonstrates the almost unlimited possibilities of modern protein engineering in gene expression, protein production and modification, as well as the design and creation of new proteins [...]
International Journal of Molecular Sciences
In the last two decades, bifunctional proteins have been created by genetic and protein engineeri... more In the last two decades, bifunctional proteins have been created by genetic and protein engineering methods to increase therapeutic effects in various diseases, including cancer. Unlike conventional small molecule or monotargeted drugs, bifunctional proteins have increased biological activity while maintaining low systemic toxicity. The recombinant anti-cancer cytokine TRAIL has shown a limited therapeutic effect in clinical trials. To enhance the efficacy of TRAIL, we designed the HRH–DR5-B fusion protein based on the DR5-selective mutant variant of TRAIL fused to the anti-angiogenic synthetic peptide HRHTKQRHTALH. Initially low expression of HRH–DR5-B was enhanced by the substitution of E. coli-optimized codons with AT-rich codons in the DNA sequence encoding the first 7 amino acid residues of the HRH peptide. However, the HRH–DR5-B degraded during purification to form two adjacent protein bands on the SDS-PAGE gel. The replacement of His by Ser at position P2 immediately after th...
Crystals, 2021
The secondary structure of horse cytochrome c with mutations in the P76GTKMIFA83 site of the Ω-lo... more The secondary structure of horse cytochrome c with mutations in the P76GTKMIFA83 site of the Ω-loop, exhibiting reduced efficiency of electron transfer, were studied. CD spectroscopy studies showed that the ordering of mutant structure increases by 3–6% compared to that of the WT molecules due to the higher content of β-structural elements. The IR spectroscopy data are consistent with the CD results and demonstrate that some α-helical elements change into β-structures, and the amount of the non-structured elements is decreased. The analysis of the 1H-NMR spectra demonstrated that cytochrome c mutants have a well-determined secondary structure with some specific features related to changes in the heme microenvironment. The observed changes in the structure of cytochrome c mutants are likely to be responsible for the decrease in the conformational mobility of the P76GTKMIFA83 sequence carrying mutations and for the decline in succinate:cytochrome c-reductase and cytochrome c-oxidase a...
Moscow University Biological Sciences Bulletin, 2016
We studied the possibility of using a broadly neutralizing anti-influenza A antibody as a module ... more We studied the possibility of using a broadly neutralizing anti-influenza A antibody as a module for the development of different protein constructs for diagnostics. For this purpose, we constructed two recombinant proteins-a Fab-fragment of the antibody and Fab-mCherry, which is a hybrid of the Fab-fragment and the mCherry fluorescent protein. Both proteins were expressed in Escherichia coli cells and purified in a functionally active state from culture medium. The antibody Fab-fragment was shown to bind all 11 tested strains of the influenza A H1N1 and H3N2 subtypes. A stronger binding was observed for group I hemagglutinins; this correlates with the immunochemical profile of the parental antibody. Comparison of the dissociation constants of complexes of the antibody Fab-fragment and Fab-mCherry with A(H1N1)/Solomon Islands/03/06 virus particles demonstrated that the attachment of the mCherry protein did not interfere with the antigen-binding properties of the antibody Fab-fragment.
Biochimie, 2019
The aim of this work was to find a minimal set of structurally stable pentapeptides, which allows... more The aim of this work was to find a minimal set of structurally stable pentapeptides, which allows forming a polypeptide chain of a required 3D structure. To search for factors that ensure structural stability of the pentapeptide, we generated peptide sequences with no more than three functional groups, based on the alanine pentapeptide AAAAA. We analyzed 44,860 structures of peptides by the molecular dynamics method and found that 1,225 pentapeptides over 80% of the simulation time were in a stable conformation. Clustering of these conformations revealed 54 topological types of conformationally stable pentapeptides. These conformations relate to different combined elements of the protein secondary structure. So, we obtained a minimal set of amino acid structures of conformationally stable pentapeptides, creating a complete set of different topologies that ensure the formation of pre-folded conformation of protein structures.
Moscow University Biological Sciences Bulletin, 2016
Molecular Biology, 2009
Mouse embryonic fibroblasts (MEF) with point mutation in somatic cytochrome C gene were generated... more Mouse embryonic fibroblasts (MEF) with point mutation in somatic cytochrome C gene were generated and characterized. It was shown that substitution of lysine for tryptophan in position 72 (K72W) decreased the proapoptotic functions of cytochrome C in response to staurosporin treatment without disrupting its respiratory functions. The presence of this mutation did not affect the pattern of cytochrome C gene expression or its localization inside the cell. These cell cultures therefore represent an interesting model for study apoptotic signaling and physiological functions of cytochrome C.
Journal of Biological Chemistry, 2011
Discovery of proteins expressed in the central nervous system sharing the three-finger structure ... more Discovery of proteins expressed in the central nervous system sharing the three-finger structure with snake ␣-neurotoxins provoked much interest to their role in brain functions. Prototoxin LYNX1, having homology both to Ly6 proteins and threefinger neurotoxins, is the first identified member of this family membrane-tethered by a GPI anchor, which considerably complicates in vitro studies. We report for the first time the NMR spatial structure for the water-soluble domain of human LYNX1 lacking a GPI anchor (ws-LYNX1) and its concentration-dependent activity on nicotinic acetylcholine receptors (nAChRs). At 5-30 M, ws-LYNX1 competed with 125 I-␣-bungarotoxin for binding to the acetylcholine-binding proteins (AChBPs) and to Torpedo nAChR. Exposure of Xenopus oocytes expressing ␣7 nAChRs to 1 M ws-LYNX1 enhanced the response to acetylcholine, but no effect was detected on ␣42 and ␣32 nAChRs. Increasing ws-LYNX1 concentration to 10 M caused a modest inhibition of these three nAChR subtypes. A common feature for ws-LYNX1 and LYNX1 is a decrease of nAChR sensitivity to high concentrations of acetylcholine. NMR and functional analysis both demonstrate that ws-LYNX1 is an appropriate model to shed light on the mechanism of LYNX1 action. Computer modeling, based on ws-LYNX1 NMR structure and AChBP x-ray structure, revealed a possible mode of ws-LYNX1 binding.
Biochemistry (Moscow), 2009
The gene for the "weak" toxin of Naja kaouthia ... more The gene for the "weak" toxin of Naja kaouthia venom was expressed in Escherichia coli. "Weak" toxin is a specific inhibitor of nicotine acetylcholine receptor, but mechanisms of interaction of similar neurotoxins with receptors are still unknown. Systems previously elaborated for neurotoxin II from venom of the cobra Naja oxiana were tested for bacterial production of "weak" toxin from N. kaouthia venom. Constructs were designed for cytoplasmic production of N. kaouthia "weak" toxin in the form of a fused polypeptide chain with thioredoxin and for secretion with the leader peptide STII. However, it became possible to obtain "weak" toxin in milligram amounts only within cytoplasmic inclusion bodies. Different approaches for refolding of the toxin were tested, and conditions for optimization of the yield of the target protein during refolding were investigated. The resulting protein was characterized by mass spectrometry and CD and NMR spectroscopy. Experiments on competitive inhibition of (125)I-labeled alpha-bungarotoxin binding to the Torpedo californica electric organ membranes containing the muscle-type nicotine acetylcholine receptor (alpha1(2)beta1gammadelta) showed the presence of biological activity of the recombinant "weak" toxin close to the activity of the natural toxin (IC(50) = 4.3 +/- 0.3 and 3.0 +/- 0.5 microM, respectively). The interaction of the recombinant toxin with alpha7 type human neuronal acetylcholine receptor transfected in the GH(4)C(1) cell line also showed the presence of activity close to that of the natural toxin (IC(50) 31 +/- 5.0 and 14.8 +/- 1.3 microM, respectively). The developed bacterial system for production of N. kaouthia venom "weak" toxin was used to obtain (15)N-labeled analog of the neurotoxin.
Biochemistry (Moscow), 2009
In recent years the development of new methods for cost-effective production of pharmaceuticals h... more In recent years the development of new methods for cost-effective production of pharmaceuticals has become increasingly important. Use of plant expression systems for recombinant protein production has a number of advantages such as inexpensive cultivation, possibility of simultaneous production of a large amount of raw material and comparatively easy storage and transportation. In addition, the technology of human protein production in plants is usually safe because there are no common pathogens. Present knowledge demonstrates that recombinant proteins expressed in plants are biologically active and can be used as pharmaceuticals [1, 2]. Earlier, we developed a system for expression of foreign proteins in Nicotiana benthamiana leaves using phytoviral vectors [3]. Application of plant viral vectors challenges such gene expression methods as use of stably transformed or transiently modified plant tissues. Due to extremely high rate of viral RNA replication, high copy number of foreign genes transcripts is achieved in cytoplasm of infected cells. Therefore, the efficiency of a viral expression system is two degrees higher comparing with stably transformed plants [1]. In addition, viral infection
Biochemical and Biophysical Research Communications, 2000
Human recombinant prothymosin ␣ (ProT␣) is known to have coil-like conformation at neutral pH; i.... more Human recombinant prothymosin ␣ (ProT␣) is known to have coil-like conformation at neutral pH; i.e., it belongs to the class of "natively unfolded" proteins. By means of circular dichroism, SAXS, and ANS fluorescence, we have investigated the effect of several divalent cations on the structure of this protein. Results of these studies are consistent with the conclusion that ProT␣ conformation is unaffected by large excess of Ca 2؉ , Mg 2؉ , Mn 2؉ , Cu 2؉ , and Ni 2؉. However, Zn 2؉ induces compaction and considerable rearrangement of the protein structure. This means that ProT␣ can specifically interact with Zn 2؉ (K D ϳ 10 ؊3 M), and such interactions induce folding of the natively unfolded protein into a compact partially folded (premolten globule-like) conformation. It is possible that these structural changes may be important for the function of this protein.
Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology, 1999
Two permuted variants of S6 ribosomal protein were obtained in direct and fusion expression syste... more Two permuted variants of S6 ribosomal protein were obtained in direct and fusion expression systems, respectively. The product of direct expression contained the extra N-terminal methionine residue. The structural properties and conformational stability of these permuteins were compared using 1-D 1 H-NMR, circular dichroism, intrinsic fluorescence, differential scanning calorimetry and resistance to urea-induced unfolding. A pronounced difference in all the parameters studied has been demonstrated. This means that the structure of recombinant protein can be sensitive to peculiarities of the expression and purification procedures, leading particularly to the presence or absence of the Met at the first position in the target protein sequence.
Moscow University Biological Sciences Bulletin, 2011
Informational structure of cytochrome c was investigated using the ANIS method (Analysis of Infor... more Informational structure of cytochrome c was investigated using the ANIS method (Analysis of Informational Structure Method). Mutant variants of cytochrome c gene were constructed on the basis of data from the ANIS method. The mutations carry substitutions reducing electron transport activity of cyto chrome c in the mitochondrial respiratory chain. These mutant genes were obtained and expressed in the bac terial system. The biological activity of the obtained cytochrome c mutant variants interacting with complexes III and IV of the respiratory chain in the system of rat liver mitoplasts.
Journal of Biological Chemistry, 2015
Background: Cobra's "three-finger" nonconventional toxin WTX allosterically modulates muscarinic ... more Background: Cobra's "three-finger" nonconventional toxin WTX allosterically modulates muscarinic receptors (mAChRs). Results: Activity of several WTX mutants was analyzed; toxin spatial structure and dynamics were determined; and complexes of toxin with M1 and M3 mAChRs were modeled. Conclusion: Flexible loop II is the major determinant for toxin binding to different mAChRs. Significance: Structural framework for rationalization of target-specific positive/negative allosteric regulation of mAChRs is provided. Weak toxin from Naja kaouthia (WTX) belongs to the group of nonconventional "three-finger" snake neurotoxins. It irreversibly inhibits nicotinic acetylcholine receptors and allosterically interacts with muscarinic acetylcholine receptors (mAChRs). Using site-directed mutagenesis, NMR spectroscopy, and computer modeling, we investigated the recombinant mutant WTX analogue (rWTX) which, compared with the native toxin, has an additional N-terminal methionine residue. In comparison with the wild-type toxin, rWTX demonstrated an altered pharmacological profile, decreased binding of orthosteric antagonist N-methylscopolamine to human M1-and M2-mAChRs, and increased antagonist binding to M3-mAChR. Positively charged arginine residues located in the flexible loop II were found to be crucial for rWTX interactions with all types of mAChR. Computer modeling suggested that the rWTX loop II protrudes to the M1-mAChR allosteric ligand-binding site blocking the entrance to the orthosteric site. In contrast, toxin interacts with M3-mAChR by loop II without penetration into the allosteric site. Data obtained provide new structural insight into the target-specific allosteric regulation of mAChRs by "three-finger" snake neurotoxins.
Biomolecules
The main aim of our work was to create a full-length bispecific antibody (BsAb) as a vehicle for ... more The main aim of our work was to create a full-length bispecific antibody (BsAb) as a vehicle for the targeted delivery of interferon-beta (IFN-β) to ErbB2+ tumor cells in the form of non-covalent complex of BsAb and IFN-β. Such a construct is a CrossMab-type BsAb, consisting of an ErbB2-recognizing trastuzumab moiety, a part of chimeric antibody to IFN-β, and human IgG1 Fc domain carrying knob-into-hole amino acid substitutions necessary for the proper assembly of bispecific molecules. The IFN-β- recognizing arm of BsAb not only forms a complex with the cytokine but neutralizes its activity, thus providing a mechanism to avoid the side effects of the systemic action of IFN-β by blocking IFN-β Interaction with cell receptors in the process of cytokine delivery to tumor sites. Enzyme sandwich immunoassay confirmed the ability of BsAb to bind to human IFN-β comparable to that of the parental chimeric mAb. The BsAb binds to the recombinant ErbB2 receptor, as well as to lysates of ErbB2+...
Biomolecules
A gene coding for a novel putative amylase, oligo-1,6-glucosidase from a psychrotrophic bacterium... more A gene coding for a novel putative amylase, oligo-1,6-glucosidase from a psychrotrophic bacterium Exiguobacterium sibiricum from Siberian permafrost soil was cloned and expressed in Escherichia coli. The amino acid sequence of the predicted protein EsOgl and its 3D model displayed several features characteristic for the cold-active enzymes while possessing an unusually high number of proline residues in the loops—a typical feature of thermophilic enzymes. The activity of the purified recombinant protein was tested with p-nitrophenyl α-D-glucopyranoside as a substrate. The enzyme displayed a plateau-shaped temperature-activity profile with the optimum at 25 °C and a pronounced activity at low temperatures (50% of maximum activity at 5 °C). To improve the thermal stability at temperatures above 40 °C, we have introduced proline residues into four positions of EsOgl by site-directed mutagenesis according to “the proline rule”. Two of the mutants, S130P and A109P demonstrated a three- a...
International Journal of Molecular Sciences
Peptide pore blockers and their fluorescent derivatives are useful molecular probes to study the ... more Peptide pore blockers and their fluorescent derivatives are useful molecular probes to study the structure and functions of the voltage-gated potassium Kv1.3 channel, which is considered as a pharmacological target in the treatment of autoimmune and neurological disorders. We present Kv1.3 fluorescent ligand, GFP–MgTx, constructed on the basis of green fluorescent protein (GFP) and margatoxin (MgTx), the peptide, which is widely used in physiological studies of Kv1.3. Expression of the fluorescent ligand in E. coli cells resulted in correctly folded and functionally active GFP–MgTx with a yield of 30 mg per 1 L of culture. Complex of GFP–MgTx with the Kv1.3 binding site is reported to have the dissociation constant of 11 ± 2 nM. GFP–MgTx as a component of an analytical system based on the hybrid KcsA–Kv1.3 channel is shown to be applicable to recognize Kv1.3 pore blockers of peptide origin and to evaluate their affinities to Kv1.3. GFP–MgTx can be used in screening and pre-selection...
Biomolecules
A key event in the cytochrome c-dependent apoptotic pathway is the permeabilization of the outer ... more A key event in the cytochrome c-dependent apoptotic pathway is the permeabilization of the outer mitochondrial membrane, resulting in the release of various apoptogenic factors, including cytochrome c, into the cytosol. It is believed that the permeabilization of the outer mitochondrial membrane can be induced by the peroxidase activity of cytochrome c in a complex with cardiolipin. Using a number of mutant variants of cytochrome c, we showed that both substitutions of Lys residues from the universal binding site for oppositely charged Glu residues and mutations leading to a decrease in the conformational mobility of the red Ω-loop in almost all cases did not affect the ability of cytochrome c to bind to cardiolipin. At the same time, the peroxidase activity of all mutant variants in a complex with cardiolipin was three to five times higher than that of the wild type. A pronounced increase in the ability to permeabilize the lipid membrane in the presence of hydrogen peroxide, as mea...
Molecules
Quite a long time ago, Oleg B. Ptitsyn put forward a hypothesis about the possible functional sig... more Quite a long time ago, Oleg B. Ptitsyn put forward a hypothesis about the possible functional significance of the molten globule (MG) state for the functioning of proteins. MG is an intermediate between the unfolded and the native state of a protein. Its experimental detection and investigation in a cell are extremely difficult. In the last decades, intensive studies have demonstrated that the MG-like state of some globular proteins arises from either their modifications or interactions with protein partners or other cell components. This review summarizes such reports. In many cases, MG was evidenced to be functionally important. Thus, the MG state is quite common for functional cellular proteins. This supports Ptitsyn’s hypothesis that some globular proteins may switch between two active states, rigid (N) and soft (MG), to work in solution or interact with partners.
Biomolecules
This Special Issue of Biomolecules demonstrates the almost unlimited possibilities of modern prot... more This Special Issue of Biomolecules demonstrates the almost unlimited possibilities of modern protein engineering in gene expression, protein production and modification, as well as the design and creation of new proteins [...]
International Journal of Molecular Sciences
In the last two decades, bifunctional proteins have been created by genetic and protein engineeri... more In the last two decades, bifunctional proteins have been created by genetic and protein engineering methods to increase therapeutic effects in various diseases, including cancer. Unlike conventional small molecule or monotargeted drugs, bifunctional proteins have increased biological activity while maintaining low systemic toxicity. The recombinant anti-cancer cytokine TRAIL has shown a limited therapeutic effect in clinical trials. To enhance the efficacy of TRAIL, we designed the HRH–DR5-B fusion protein based on the DR5-selective mutant variant of TRAIL fused to the anti-angiogenic synthetic peptide HRHTKQRHTALH. Initially low expression of HRH–DR5-B was enhanced by the substitution of E. coli-optimized codons with AT-rich codons in the DNA sequence encoding the first 7 amino acid residues of the HRH peptide. However, the HRH–DR5-B degraded during purification to form two adjacent protein bands on the SDS-PAGE gel. The replacement of His by Ser at position P2 immediately after th...
Crystals, 2021
The secondary structure of horse cytochrome c with mutations in the P76GTKMIFA83 site of the Ω-lo... more The secondary structure of horse cytochrome c with mutations in the P76GTKMIFA83 site of the Ω-loop, exhibiting reduced efficiency of electron transfer, were studied. CD spectroscopy studies showed that the ordering of mutant structure increases by 3–6% compared to that of the WT molecules due to the higher content of β-structural elements. The IR spectroscopy data are consistent with the CD results and demonstrate that some α-helical elements change into β-structures, and the amount of the non-structured elements is decreased. The analysis of the 1H-NMR spectra demonstrated that cytochrome c mutants have a well-determined secondary structure with some specific features related to changes in the heme microenvironment. The observed changes in the structure of cytochrome c mutants are likely to be responsible for the decrease in the conformational mobility of the P76GTKMIFA83 sequence carrying mutations and for the decline in succinate:cytochrome c-reductase and cytochrome c-oxidase a...
Moscow University Biological Sciences Bulletin, 2016
We studied the possibility of using a broadly neutralizing anti-influenza A antibody as a module ... more We studied the possibility of using a broadly neutralizing anti-influenza A antibody as a module for the development of different protein constructs for diagnostics. For this purpose, we constructed two recombinant proteins-a Fab-fragment of the antibody and Fab-mCherry, which is a hybrid of the Fab-fragment and the mCherry fluorescent protein. Both proteins were expressed in Escherichia coli cells and purified in a functionally active state from culture medium. The antibody Fab-fragment was shown to bind all 11 tested strains of the influenza A H1N1 and H3N2 subtypes. A stronger binding was observed for group I hemagglutinins; this correlates with the immunochemical profile of the parental antibody. Comparison of the dissociation constants of complexes of the antibody Fab-fragment and Fab-mCherry with A(H1N1)/Solomon Islands/03/06 virus particles demonstrated that the attachment of the mCherry protein did not interfere with the antigen-binding properties of the antibody Fab-fragment.
Biochimie, 2019
The aim of this work was to find a minimal set of structurally stable pentapeptides, which allows... more The aim of this work was to find a minimal set of structurally stable pentapeptides, which allows forming a polypeptide chain of a required 3D structure. To search for factors that ensure structural stability of the pentapeptide, we generated peptide sequences with no more than three functional groups, based on the alanine pentapeptide AAAAA. We analyzed 44,860 structures of peptides by the molecular dynamics method and found that 1,225 pentapeptides over 80% of the simulation time were in a stable conformation. Clustering of these conformations revealed 54 topological types of conformationally stable pentapeptides. These conformations relate to different combined elements of the protein secondary structure. So, we obtained a minimal set of amino acid structures of conformationally stable pentapeptides, creating a complete set of different topologies that ensure the formation of pre-folded conformation of protein structures.
Moscow University Biological Sciences Bulletin, 2016
Molecular Biology, 2009
Mouse embryonic fibroblasts (MEF) with point mutation in somatic cytochrome C gene were generated... more Mouse embryonic fibroblasts (MEF) with point mutation in somatic cytochrome C gene were generated and characterized. It was shown that substitution of lysine for tryptophan in position 72 (K72W) decreased the proapoptotic functions of cytochrome C in response to staurosporin treatment without disrupting its respiratory functions. The presence of this mutation did not affect the pattern of cytochrome C gene expression or its localization inside the cell. These cell cultures therefore represent an interesting model for study apoptotic signaling and physiological functions of cytochrome C.
Journal of Biological Chemistry, 2011
Discovery of proteins expressed in the central nervous system sharing the three-finger structure ... more Discovery of proteins expressed in the central nervous system sharing the three-finger structure with snake ␣-neurotoxins provoked much interest to their role in brain functions. Prototoxin LYNX1, having homology both to Ly6 proteins and threefinger neurotoxins, is the first identified member of this family membrane-tethered by a GPI anchor, which considerably complicates in vitro studies. We report for the first time the NMR spatial structure for the water-soluble domain of human LYNX1 lacking a GPI anchor (ws-LYNX1) and its concentration-dependent activity on nicotinic acetylcholine receptors (nAChRs). At 5-30 M, ws-LYNX1 competed with 125 I-␣-bungarotoxin for binding to the acetylcholine-binding proteins (AChBPs) and to Torpedo nAChR. Exposure of Xenopus oocytes expressing ␣7 nAChRs to 1 M ws-LYNX1 enhanced the response to acetylcholine, but no effect was detected on ␣42 and ␣32 nAChRs. Increasing ws-LYNX1 concentration to 10 M caused a modest inhibition of these three nAChR subtypes. A common feature for ws-LYNX1 and LYNX1 is a decrease of nAChR sensitivity to high concentrations of acetylcholine. NMR and functional analysis both demonstrate that ws-LYNX1 is an appropriate model to shed light on the mechanism of LYNX1 action. Computer modeling, based on ws-LYNX1 NMR structure and AChBP x-ray structure, revealed a possible mode of ws-LYNX1 binding.
Biochemistry (Moscow), 2009
The gene for the "weak" toxin of Naja kaouthia ... more The gene for the "weak" toxin of Naja kaouthia venom was expressed in Escherichia coli. "Weak" toxin is a specific inhibitor of nicotine acetylcholine receptor, but mechanisms of interaction of similar neurotoxins with receptors are still unknown. Systems previously elaborated for neurotoxin II from venom of the cobra Naja oxiana were tested for bacterial production of "weak" toxin from N. kaouthia venom. Constructs were designed for cytoplasmic production of N. kaouthia "weak" toxin in the form of a fused polypeptide chain with thioredoxin and for secretion with the leader peptide STII. However, it became possible to obtain "weak" toxin in milligram amounts only within cytoplasmic inclusion bodies. Different approaches for refolding of the toxin were tested, and conditions for optimization of the yield of the target protein during refolding were investigated. The resulting protein was characterized by mass spectrometry and CD and NMR spectroscopy. Experiments on competitive inhibition of (125)I-labeled alpha-bungarotoxin binding to the Torpedo californica electric organ membranes containing the muscle-type nicotine acetylcholine receptor (alpha1(2)beta1gammadelta) showed the presence of biological activity of the recombinant "weak" toxin close to the activity of the natural toxin (IC(50) = 4.3 +/- 0.3 and 3.0 +/- 0.5 microM, respectively). The interaction of the recombinant toxin with alpha7 type human neuronal acetylcholine receptor transfected in the GH(4)C(1) cell line also showed the presence of activity close to that of the natural toxin (IC(50) 31 +/- 5.0 and 14.8 +/- 1.3 microM, respectively). The developed bacterial system for production of N. kaouthia venom "weak" toxin was used to obtain (15)N-labeled analog of the neurotoxin.
Biochemistry (Moscow), 2009
In recent years the development of new methods for cost-effective production of pharmaceuticals h... more In recent years the development of new methods for cost-effective production of pharmaceuticals has become increasingly important. Use of plant expression systems for recombinant protein production has a number of advantages such as inexpensive cultivation, possibility of simultaneous production of a large amount of raw material and comparatively easy storage and transportation. In addition, the technology of human protein production in plants is usually safe because there are no common pathogens. Present knowledge demonstrates that recombinant proteins expressed in plants are biologically active and can be used as pharmaceuticals [1, 2]. Earlier, we developed a system for expression of foreign proteins in Nicotiana benthamiana leaves using phytoviral vectors [3]. Application of plant viral vectors challenges such gene expression methods as use of stably transformed or transiently modified plant tissues. Due to extremely high rate of viral RNA replication, high copy number of foreign genes transcripts is achieved in cytoplasm of infected cells. Therefore, the efficiency of a viral expression system is two degrees higher comparing with stably transformed plants [1]. In addition, viral infection
Biochemical and Biophysical Research Communications, 2000
Human recombinant prothymosin ␣ (ProT␣) is known to have coil-like conformation at neutral pH; i.... more Human recombinant prothymosin ␣ (ProT␣) is known to have coil-like conformation at neutral pH; i.e., it belongs to the class of "natively unfolded" proteins. By means of circular dichroism, SAXS, and ANS fluorescence, we have investigated the effect of several divalent cations on the structure of this protein. Results of these studies are consistent with the conclusion that ProT␣ conformation is unaffected by large excess of Ca 2؉ , Mg 2؉ , Mn 2؉ , Cu 2؉ , and Ni 2؉. However, Zn 2؉ induces compaction and considerable rearrangement of the protein structure. This means that ProT␣ can specifically interact with Zn 2؉ (K D ϳ 10 ؊3 M), and such interactions induce folding of the natively unfolded protein into a compact partially folded (premolten globule-like) conformation. It is possible that these structural changes may be important for the function of this protein.
Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology, 1999
Two permuted variants of S6 ribosomal protein were obtained in direct and fusion expression syste... more Two permuted variants of S6 ribosomal protein were obtained in direct and fusion expression systems, respectively. The product of direct expression contained the extra N-terminal methionine residue. The structural properties and conformational stability of these permuteins were compared using 1-D 1 H-NMR, circular dichroism, intrinsic fluorescence, differential scanning calorimetry and resistance to urea-induced unfolding. A pronounced difference in all the parameters studied has been demonstrated. This means that the structure of recombinant protein can be sensitive to peculiarities of the expression and purification procedures, leading particularly to the presence or absence of the Met at the first position in the target protein sequence.
Moscow University Biological Sciences Bulletin, 2011
Informational structure of cytochrome c was investigated using the ANIS method (Analysis of Infor... more Informational structure of cytochrome c was investigated using the ANIS method (Analysis of Informational Structure Method). Mutant variants of cytochrome c gene were constructed on the basis of data from the ANIS method. The mutations carry substitutions reducing electron transport activity of cyto chrome c in the mitochondrial respiratory chain. These mutant genes were obtained and expressed in the bac terial system. The biological activity of the obtained cytochrome c mutant variants interacting with complexes III and IV of the respiratory chain in the system of rat liver mitoplasts.
Journal of Biological Chemistry, 2015
Background: Cobra's "three-finger" nonconventional toxin WTX allosterically modulates muscarinic ... more Background: Cobra's "three-finger" nonconventional toxin WTX allosterically modulates muscarinic receptors (mAChRs). Results: Activity of several WTX mutants was analyzed; toxin spatial structure and dynamics were determined; and complexes of toxin with M1 and M3 mAChRs were modeled. Conclusion: Flexible loop II is the major determinant for toxin binding to different mAChRs. Significance: Structural framework for rationalization of target-specific positive/negative allosteric regulation of mAChRs is provided. Weak toxin from Naja kaouthia (WTX) belongs to the group of nonconventional "three-finger" snake neurotoxins. It irreversibly inhibits nicotinic acetylcholine receptors and allosterically interacts with muscarinic acetylcholine receptors (mAChRs). Using site-directed mutagenesis, NMR spectroscopy, and computer modeling, we investigated the recombinant mutant WTX analogue (rWTX) which, compared with the native toxin, has an additional N-terminal methionine residue. In comparison with the wild-type toxin, rWTX demonstrated an altered pharmacological profile, decreased binding of orthosteric antagonist N-methylscopolamine to human M1-and M2-mAChRs, and increased antagonist binding to M3-mAChR. Positively charged arginine residues located in the flexible loop II were found to be crucial for rWTX interactions with all types of mAChR. Computer modeling suggested that the rWTX loop II protrudes to the M1-mAChR allosteric ligand-binding site blocking the entrance to the orthosteric site. In contrast, toxin interacts with M3-mAChR by loop II without penetration into the allosteric site. Data obtained provide new structural insight into the target-specific allosteric regulation of mAChRs by "three-finger" snake neurotoxins.
Biomolecules
The main aim of our work was to create a full-length bispecific antibody (BsAb) as a vehicle for ... more The main aim of our work was to create a full-length bispecific antibody (BsAb) as a vehicle for the targeted delivery of interferon-beta (IFN-β) to ErbB2+ tumor cells in the form of non-covalent complex of BsAb and IFN-β. Such a construct is a CrossMab-type BsAb, consisting of an ErbB2-recognizing trastuzumab moiety, a part of chimeric antibody to IFN-β, and human IgG1 Fc domain carrying knob-into-hole amino acid substitutions necessary for the proper assembly of bispecific molecules. The IFN-β- recognizing arm of BsAb not only forms a complex with the cytokine but neutralizes its activity, thus providing a mechanism to avoid the side effects of the systemic action of IFN-β by blocking IFN-β Interaction with cell receptors in the process of cytokine delivery to tumor sites. Enzyme sandwich immunoassay confirmed the ability of BsAb to bind to human IFN-β comparable to that of the parental chimeric mAb. The BsAb binds to the recombinant ErbB2 receptor, as well as to lysates of ErbB2+...
Biomolecules
A gene coding for a novel putative amylase, oligo-1,6-glucosidase from a psychrotrophic bacterium... more A gene coding for a novel putative amylase, oligo-1,6-glucosidase from a psychrotrophic bacterium Exiguobacterium sibiricum from Siberian permafrost soil was cloned and expressed in Escherichia coli. The amino acid sequence of the predicted protein EsOgl and its 3D model displayed several features characteristic for the cold-active enzymes while possessing an unusually high number of proline residues in the loops—a typical feature of thermophilic enzymes. The activity of the purified recombinant protein was tested with p-nitrophenyl α-D-glucopyranoside as a substrate. The enzyme displayed a plateau-shaped temperature-activity profile with the optimum at 25 °C and a pronounced activity at low temperatures (50% of maximum activity at 5 °C). To improve the thermal stability at temperatures above 40 °C, we have introduced proline residues into four positions of EsOgl by site-directed mutagenesis according to “the proline rule”. Two of the mutants, S130P and A109P demonstrated a three- a...