Laboratory of optical microscopy and spectroscopy of biomolecules

Department of structural biology

Head: Alexey Feofanov, D.Sc, assistant professor
avfeofanov@yandex.ru+7(495)336-64-55

www.ibch.ru/structure/groups/optics

Circular dichroism, membrane and membrane-active proteins, microspectroscopy, optical microscopy, efrin receptors, fluorescence, photosensitizers

The Laboratory studies biologically active molecules, membranous and membrane-active proteins and develops new methods of optical microscopy and spectroscopy to support these studies. Developed methods are used to study the action mechanisms and structural-functional interrelationships of investigated molecules at different levels of structural organization: molecular, cellular and tissue.

The methods of microscopy of single-molecules and their complexes are developed in the Laboratory using the unique scientific equipment. It includes an installation for one-photon and multiphoton 4Pi superresolution microscopy and an installation for fluorescence microscopy based on the effect of total internal reflection. Both installations have got a high sensitivity, allowing registering fluorescent signals even from single molecules.

Experimental installation for confocal laser microspectroscopy was created in the Laboratory. Also, the method of confocal microspectroscopy and reconstruction of spectral images (COMIRSI) was developed that provided possibility to identify and study molecular interactions of biologically active compounds in living cells with a three-dimensional submicron spatial resolution. The experimental installation and the COMIRSI method are widely used  in the development, scientific and clinical studies of new Russian photosensitizers for photodynamic cancer therapy, as well as in the studies of membrane and membrane-active proteins.

The Laboratory is open for cooperation with the departments of the Institute and other scientific organizations.

The development of analytical systems based on hybrid ion channels for the search and study of potassium channel blockers is conducted jointly with the Group of nanobioengineering. A search and study of potassium channel blockers in scorpion venoms are carried out in collaboration with the Molecular Instruments for Neurobiology Group. The functional properties of toxins from snake venom are studied together with the Laboratory of Molecular Toxinology.

Also, the Laboratory conducts joint studies with a number of other scientific organizations. Study of photosensitizers for photodynamic anti-cancer and antimicrobial therapy and optimization of their properties is carried out in cooperation with the Moscow Technological University, the P.A. Herzen Moscow Oncology Research Institute and the State Scientific Center "NIOPIK". Studies of structural rearrangements in nucleosomes under the influence of various nuclear proteins using the methods of fluorescence microscopy of single molecules and their complexes are carried out together with the Bioengineering Department, the Laboratory for the Regulation of Transcription and Replication of the Biological Faculty of Moscow State University and colleagues from Fox Chase Cancer Center (USA). The structure optimization of conjugates of boron-containing nanoparticles and natural porphyrins is conducted jointly with the Moscow Technological University and INEOS RAS.

The laboratory collaborates with foreign scientific organizations. Biomedical nanosensors for the diagnosis and treatment of breast cancer are being developed together with the University of Tours (France) and the Rhine-Waal University of Applied Sciences (Germany).

The Laboratory employees teach the basics of optical microscopy to students of the ESC of the IBCh RAS and the Department of Bioengineering of the Biological Faculty of the Moscow State University. Each year, several bachelor's and master's works as well as scientific research of graduate students and young scientists are conducted. under the supervision of the Laboratory employees

The Laboratory of optical microscopy and spectroscopy of biomolecules was established in 2005. It is a part of the Department of Structural Biology, which arose as a result of reorganization of the Laboratory of Instrumental Methods of Analysis. This reorganization was  conducted due to the initiative of professor A.S. Arsenyev, who is the current head of the Department.

A three-dimensional confocal image of HEK293 cells in which a stable expression of the EphA2 receptor of EphA2 fused to a cyan fluorescent protein (shown in green) is achieved, together with EphA2 fused to a yellow fluorescent protein (shown in red). The coincidence of the two structures localization is shown in yellow. The cell model is used to study the mechanisms of activation and dimerization of Efrin receptors using the method of resonant fluorescence energy transfer (FRET).

 

  • Cellular bioengineering systems and methods of their applications using fluorescence microscopy have been developed. They are used to study pore blockers of voltage-gated potassium channels and to search for peptide blockers in natural venoms.
  • Using fluorescent proteins and scorpion toxins,  the high-affinity conjugates were designed that interact selectively with Kv1 channels and can be used for their visualization.   
  • New high-affinity channel blockers of Kv1.x (x = 1-3.6) were discovered in scorpion venoms and characterized by activity.
  • Recombinant peptide-blockers of Kv-channels having improved selectivity were created.  Molecular models of peptide-blockers complexes with Kv-channels were constructed.; Interaction interfaces and amino acid residues affecting the force and selectivity of interactions were described.
  • Methods for studying single molecules and their complexes in the free diffusion regime  and in the immobilized state have been developed based on Forster resonance energy transfer microscopy.  Applicability of these methods for investigation of structural changes of nucleosomal DNA in complexes with different protein factors was demonstrated.  
  • Structures of new conjugates of boron-containing nanoparticles and natural porphyrins have been optimized that provided delivery of more than 1 billion boron atoms per cancer cell. In this way, applicability of new nanoconjugates for boron-neutron capture therapy (BNCT) was provided. Nanoconjugates are active photosensitizers and cause photoinduced death of tumor cells at nanomolar concentrations. New conjugates are perspective multifunctional agents for photodynamic therapy, BNСT and fluorescent cancer diagnostics.
  • It has been established that the key structural element determining high accumulation of chlorin e6 conjugates with cobalt bis(dicarbolide) nanoparticle in cancer cells is the amino-polyalkyl-amine linker that connects a porphyrin chromophore to nanoparticle. Photocytotoxicity of conjugates is based on photoinduced lipid peroxidation, lysosomes permeabilization, and proteases activation in cytoplasm of cells.
NamePositionContacts
Alexey Feofanov, D.Sc, assistant professorHead of lab.avfeofanov@yandex.ru+7(495)336-64-55
Anastasia Efremenkor. f.aefr@mail.ru
Anastasia Ignatovar. f.aignatova_83@mail.ru+7(495)336-17-66
Kseniya Markvicheva, Ph.D.j. r. f.markvicheva@gmail.com+7()
Anna Feofanovat. q. - lab. as.+7(495)336-64-55

Former members:

Marija Astapova, Ph.D.s. r. f.ast-mariya@yandex.ru
George Sharonov, Ph.D.s. r. f.sharonov@gmail.com

All publications (show selected)

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Alexey Feofanov

Mechanisms of anticancer action of curaxins

In collaboration with Group of nanobioengineering

Data of spFRET analysis support  the hypothesis that  anticancer drug curaxin, namely, its  CBL0137 derivative, can  affect long-distance enhancer-promoter communication (EPC) in chromatin by disrupting nucleosome structure or affecting the structure and dynamics of the linker DNA supporting efficient EPC (Kantidze et al., Nat Commun., 2019,10(1):1441). The data indicate also that CBL0137 attracts human FACT (protein factor that FAcilitates Chromatin Transcription) to nucleosomes, mediates hFACT-induced scaled, partially reversible nucleosome unfolding (or uncoiling of the nucleosomal DNA) and traps hFACT on nucleosomes. This curaxin-dependent FACT trapping can be a reason of hFACT redistribution from the transcribed chromatin regions to other genomic loci and contribute to the anticancer action of curaxins (Chang et al. Science Advances, 2018, 4 (11), eaav2131).

The studies were performed jointly with the specialists from the Institute of Gene Biology RAS (Kantidze O.L., Luzhin A.V., Golov A.K., Velichko A.K.),   Biology Faculty of Lomonosov Moscow State University (Valieva M.E., Lyubitelev A.V., Razin S.V.), Fox Chase Cancer Center, USA (Nizovtseva E.V., Studitsky V.M., Kulaeva O.I., Chang H.-W.), Roswell Park Comprehensive Cancer Center, USA (Gurova K.V., Safina A., Wang J.,), Eunice Kennedy Shriver National Institute for Child Health and Human Development, USA (Chereji R.V.), Rutgers University, USA (Morozov A.V.).

Publications

  1. Kantidze OL, Luzhin AV, Nizovtseva EV, Safina A, Valieva ME, Golov AK, Velichko AK, Lyubitelev AV, Feofanov AV, Gurova KV, Studitsky VM, Razin SV (2019). The anti-cancer drugs curaxins target spatial genome organization. Nat Commun 10 (1), 1441
  2. Chang HW, Valieva ME, Safina A, Chereji RV, Wang J, Kulaeva OI, Morozov AV, Kirpichnikov MP, Feofanov AV, Gurova KV, Studitsky VM (2018). Mechanism of FACT removal from transcribed genes by anticancer drugs curaxins. Sci Adv 4 (11), eaav2131

MeKTx11-1, Kv1.2 channel –specific peptide blocker from the M.eupeus scorpion venom: structural basis of selectivity

In collaboration with Laboratory of biomolecular modeling,  Group of in silico analysis of membrane proteins structure,  Group of nanobioengineering,  Laboratory of Molecular Instruments for Neurobiology

A.V. Feofanov (Laboratory of optical microscopy and spectroscopy of biomolecules), О.V. Nekrasova, K.S.Kudryashova (Group of nanobioengineering, Bioengineering department), A.A. Vassilevski, A.I. Kuzmenkov, A.M. Gigolaev (Laboratory of molecular instruments for neurobiology), A.O. Chugunov, V.M. Tabakmakher, R.G. Efremov (Group of in silico analysis of membrane proteins structure, Laboratory of biomolecular modeling).

A unique high-affinity and highly selective peptide blocker of Kv1.2 channel, MeKTx11-1, from the scorpion venom Mesobuthus eupeus was studied. Peptide MeKTx11-1 and its mutant forms were produced in a recombinant form, and their receptor-binding activity was studied against a panel of Kv1-channels. Molecular modeling of interaction of these peptides with Kv1.2 channel was carried out, and key structural elements of the interactions were determined. Peptide MeKTx11-1 may be used as a novel efficient molecular tool in neurobiology to identify and study the activity of Kv1.2 channel in the presence of different isoforms of Kv1-channels.

In collaboration with S.Peigneur and J.Tytgat fromUniversity of Leuven, Belgium and A.F. Fradkov from Evrogen JSC.

Mammalian three-finger proteins protect against cancer

In collaboration with Laboratory of structural biology of ion channels,  Laboratory of bioengineering of neuromodulators and neuroreceptors

Efremenko A.V., Sharonov G.V., Feofanov A.V. (Laboratory of optical microscopy and spectroscopy of biomolecules), Lyukmanova E.N., Bychkov M.L., Shulepko M.A., Kulbatskii D.S., Dolgikh D.A., Kirpichnikov M.P. (Group of bioengineering of neuromodulators and neuroreceptors), Shenkarev Z.O. (Group of structural biology of ion channels).

The human secreted protein SLURP-1, which is expressed in epithelial cells and controls their proliferation and migration, has been found to inhibit the growth of epithelial cancer cells. The effect of SLURP-1 on cancer cells is characterized by a positive feedback: exogenous (recombinant) SLURP-1 binds to α7 nicotinic acetylcholine receptors on the cell membrane and triggers a cascade of signals that activates secretion of endogenous SLURP-1 from intracellular depot, quickly increasing its concentration in the intercellular space and enhancing antiproliferative action. Concentrations of SLURP-1, which suppress the growth of cancer cells, do not affect the growth of normal cells.

MeKTx11-1, Kv1.2 channel –specific peptide blocker from the M.eupeus scorpion venom: structural basis of selectivity

In collaboration with Group of in silico analysis of membrane proteins structure,  Laboratory of Molecular Instruments for Neurobiology,  Group of nanobioengineering

Оksana V. Nekrasova, K.S.Kudryashova (Group of nanobioengineering, Bioengineering department), A.A. Vassilevski, A.I. Kuzmenkov, A.M. Gigolaev (Laboratory of molecular instruments for neurobiology), A.O. Chugunov, V.M. Tabakmakher, R.G. Efremov (Group of in silico analysis of membrane proteins structure, Laboratory of biomolecular modeling), A.V. Feofanov (Laboratory of optical microscopy and spectroscopy of biomolecules).

A unique high-affinity and highly selective peptide blocker of Kv1.2 channel, MeKTx11-1, from the scorpion venom Mesobuthus eupeus was studied. Peptide MeKTx11-1 and its mutant forms were produced in a recombinant form, and their receptor-binding activity was studied against a panel of Kv1-channels. Molecular modeling of interaction of these peptides with Kv1.2 channel was carried out, and key structural elements of the interactions were determined. Peptide MeKTx11-1 may be used as a novel efficient molecular tool in neurobiology to identify and study the activity of Kv1.2 channel in the presence of different isoforms of Kv1-channels.

In collaboration with S.Peigneur and J.Tytgat fromUniversity of Leuven, Belgium and A.F. Fradkov from Evrogen JSC.

Motif-based design of bioactive peptides

In collaboration with Laboratory of ligand-receptor interactions,  Laboratory of biomolecular modeling

Engineering antimicrobial peptide with a low hemolytic activity via combination of motifs of spider venom peptides. Using coarse-grained Molecular Dynamics, the depth of penetration of parent spider venom peptides (Ltc1, Oxt 4a) in model erythrocyte membrane was estimated. The artificial peptide (P5) is formed of fragments with the low depth of penetration (encircled - see Fig.), or penetrating deeply, and thus hemolytic (enclosed in ellipse). Hydrophobicity of the latter peptide was decreased via L/K mutation.

Mode of selective action of Kv1.2 potassium channel blocker

In collaboration with Group of in silico analysis of membrane proteins structure,  Group of nanobioengineering,  Laboratory of Molecular Instruments for Neurobiology

Scorpion venom is rich in peptide blockers of voltage-gated potassium channels (KV), and we have reflected this diversity previously in Kalium, a database dedicated to such peptides. A high-affinity and selective blocker of KV1.2 channels, characteristic of the human central nervous system, was obtained from the venom of the scorpion Mesobuthus eupeus. Using molecular modeling and site-directed mutagenesis, the mechanism of selective interaction between the toxin and channels was investigated.

KV1.2 Сhannel-Specific Blocker from Scorpion Venom: Structural Basis of Selectivity

In collaboration with Group of nanobioengineering,  Laboratory of Molecular Instruments for Neurobiology,  Group of in silico analysis of membrane proteins structure

Laboratory of Molecular Instruments for Neurobiology is known for systematic study of Arthropods’ venoms and derived peptides that specifically target various ion channels. Scorpions’ venom is abundant with potassium channels (Kv) blockers, and this diversity was described in previously released in Kalium database.

In cooperation with Laboratory of optical microscopy and spectroscopy of biomolecules and Group of nanobioengineering an unique screening system permitted identification in the Mesobuthus eupeus scorpion venom of Kv1.2 blocker: peptide MeKTx11-1 binging with high affinity (IC50 ≈0,2 nM) and specificity (effect on Kv1.1, 1.3 and 1.6 emerges at >100-fold higher concentrations). This peptide differs from the related MeKTx11-3 by just two residues, possessing substantially lower Kv1.2-specificity.

Finally, Group of in silico analysis of membrane proteins structure conducted a molecular modeling study of these two peptides interacting with Kv1.2 channel, immersed into an explicit lipid bilayer. This study uncovered mechanism of selective action of MeKTx11-1 peptide. The developed analysis technique will be of use for future design of selective ligands of Kv and other channels, which may be applied in fundamental studies of molecular basis of nervous system function and as drugs prototypes.

The human secreted protein SLURP-1, which is expressed in epithelial cells and controls their proliferation and migration, has been found to inhibit the growth of epithelial cancer cells.

The effect of SLURP-1 on cancer cells is characterized by a positive feedback: exogenous (recombinant) SLURP-1 binds to α7 nicotinic acetylcholine receptors on the cell membrane and triggers a cascade of signals that activates secretion of endogenous SLURP-1 from intracellular depot, quickly increasing its concentration in the intercellular space and enhancing antiproliferative action.

Concentrations of SLURP-1, which suppress the growth of cancer cells, do not affect the growth of normal cells.

Internalization mechanisms and intracellular distribution features of magnetic nanoparticles functionalized with folic acid

Ignatova A.A., Feofanov A.V.

Theranostics-oriented fluorescently-labeled iron oxide nanoparticles coated with polyethylene glycol and functionalized with folic acid effectively accumulate in HeLa cervix carcinoma cells having a high level of membrane folate receptors.

Penetration of nanoparticles into HeLa cells occurs primarily by clathrin-dependent endocytosis with a weak participation of caveolin-mediated endocytosis and ends with their accumulation in lysosomes.

This work was performed in collaboration with Shebanova A. (Biological Faculty, Lomonosov Moscow State University), Allard-Vannier E., Hervé-Aubert K., Kaaki K., Blondy T., Saboungi M.L., Chourpa I.(EA 6295 Nanomédicaments et Nanosondes, Université F. Rabelais de Tours, Tours, France).

Publications

  1. Allard-Vannier E, Hervé-Aubert K, Kaaki K, Blondy T, Shebanova A, Shaitan KV, Ignatova AA, Saboungi ML, Feofanov AV, Chourpa I (2017). Folic acid-capped PEGylated magnetic nanoparticles enter cancer cells mostly via clathrin-dependent endocytosis. BIOCHIM BIOPHYS ACTA 1861 (6), 1578–1586

AN EFFICIENT METHOD FOR PRODUCTION OF RECOMBINANT α-КТХ PEPTIDES – THE BLOCKERS OF POTASSIUM CHANNELS

In collaboration with Group of nanobioengineering

O.V.Nekrasova, K.S.Kudryashova, S.A.Yakimov, M.P.Kirpichnikov

A.V.Feofanov

A bioengineering method for production of peptide blockers of potassium Kv1 channels has been developed that provides:

  • high yield of the target peptides (12-22 mg/l culture);
  • retaining the native amino acid sequence of α-КТх peptides;
  • high yield of the renatured form of the peptides with correctly formed three and four disulfide bonds;
  • simple and reliable procedure of peptide isolation and purification.

The recombinant peptides of the α-KTx family obtained by this method have the activity of the natural blockers. High affinity potassium channel blockers from scorpion venom are widely used to study the structure and function of the channels and have a promising medical significance.

An efficient method for production of recombinant α-КТх peptides – the blockers of potassium channels

In collaboration with Group of nanobioengineering

A bioengineering method for production of peptide blockers of potassium Kv1 channels has been developed that provides: high yield of the target peptides (12-22 mg/l culture); retaining the native amino acid sequence of α-КТх peptides; high yield of the renatured form of the peptides with correctly formed three and four disulfide bonds; simple and reliable procedure of peptide isolation and purification. The recombinant peptides of the α-KTx family obtained by this method have the activity of the natural blockers. High affinity potassium channel blockers from scorpion venom are widely used to study the structure and function of the channels and have a promising medical value.

Development of integrated transcriptomic and proteomic approach to search for blockers of potassium channels in animal venoms

Authors: 

Kuzmenkov A.I. , Vassilevski A.A., Grishin Eu.V.

Department of molecular neurobiology

Kudryashova K.S., Nekrasova O.V., Kirpichnikov M.P.  

Bioengineering Department  

Feofanov A.V.  

Laboratory of optical microscopy and spectroscopy of biomolecules

Annotation: 

An original approach was developed to search for new ligands of potassium channels. It combines the bioengineering cellular test system and transcriptomic and proteomic analysis of animal venoms. Using this approach eight high-affinity peptide blockers of voltage-gated potassium channel Kv1.1 (including five new peptides) were found in the venom of the scorpion Mesobuthus eupeus. The proposed approach is a versatile and effective tool for directed search for  blockers of potassium channels in natural venoms.