Projects

1. Project: Comparison of bacterial FMN-dependent WrbA and eukaryotic FAD dependent oxidoreductases

Registered students (1st project): Jagat Rathod , Tursun Mahir
Registered students (2nd project): Alena Famina
Accepted students: Tursun Mahir

Project´s aim:

Biochemical and structural insight into the functions of the E. coli protein WrbA, the founding member of a widespread and highly conserved family shows that WrbA is structurally and functionally related to both bacterial FMN-dependent flavodoxins and eukaryotic FAD dependent oxidoreductases. Within the project the intern will work on biochemical and biophysical results to illuminate structure and function in the WrbA family of stress-defense proteins, using techniques such as enzyme assays, protein expression and purification, limited proteolysis, as well as isothermal titration calorimetry (ITC).

Results: Comparison of kinetic properties of bacterial FMN-dependent WrbA and the eukaryotic FAD dependent oxidoreductase DT-diaphorase in depandance of the dimer-tetramer equilibrium

Short annotation (EN):

Biochemical and structural insight into the functions of the E. coli protein WrbA, the founding member of a widespread and highly conserved family shows that WrbA is structurally and functionally related to both bacterial FMN-dependent flavodoxins and eukaryotic FAD dependent oxidoreductases. The structure of E. coli WrbA suggests new hypotheses about structural and functional evolutionary relationships within and between these two classes. The expected significance of the proposed work derives from its aim to create a comparative framework for understanding structure and function in these proteins. Within the project the intern will work on biochemical and biophysical results to illuminate structure and function in the WrbA family of stress-defense proteins, using techniques such as enzyme assays, protein expression and purification, limited proteolysis, as well as isothermal titration calorimetry (ITC).

2. Project : Modeling interactions in and between biomolecules and complex biologically relevant systems

Registered students (1st project): Sergey Golenchenko , Alena Famina
Registered students (2nd project): TILAK Gupta , Bahareh Shakiba , Sandeep C
Accepted students: Famina Alena
Final presentation: Famina Alena - Modeling interactions in and between biomolecules and complex biologically relevant Systems

Project´s aim:

During the project the intern gets an introduction to computational methods for building and investigation of biological systems and will be able to analyze dynamical changes in systems and learn how to interprete generated data. You will use special software for modeling biological systems and carrying out molecular dynamics simulations (Yasara, Gromacs, VMD…), partly using massive parallel calculations on a beowulf-computer cluster. The underlying experimental basis of all structural information comes from protein crystallography done in house. Our model systems are biological “hot topic” systems, like Type I restriction-modification enzymes EcoR124I and EcoAI, that act as conventional adenine methylases on hemi-methylated DNAs, but unmethylated recognition targets induce them to translocate thousands of basepairs before cleaving distant sites nonspecifically using a so called RecA-like ATPase motor, able to translocate double-stranded DNA. Our laboratory published the first crystal structure of the motor subunit (pdb entry 2w00, Lapkouski et al, 2009) in Nature Structural & Molecular Biology, responsible for translocation and cleavage. This structure suggests how the pentameric translocating complex is assembled and provides a structural framework for duplex DNA translocation by RecA-like ATPase motors. Verification and analysis of calculations is done in close collaboration with the molecular biology and biochemistry team within our group.

Results: Computational predictions about the molecular mechanism of enzyme activities and their mutual interactions in Type I restriction modifaction systems

Short annotation (EN):

Modeling interactions in and between biomolecules and complex biologically relevant systems . The intern will mainly work with computational tools on data, that are produced by experimentalists in house. After getting intense training during the first week, she/he will be responsible for a sub-project that leads to a clear outcome/prediction that can be experimentally verified. The intern will also get an insight how the experimental data, like the X-ray structures, are generated and will collaborate directly with the scientists working on that.

Students should hold a bachelor in biochemistry, biophysics or similar. The interest of the applicant should be in life sciences, the understanding of biologically relevant systems, and how we can understand them by computational modeling. This involves a good background in physics and mathematics, and a good relation to computers as a scientific tool.

3. Project : Recombinant protein production of a restriction-modification enzyme Type I in a fermentor.

Registered students (1st project): Ramona-Elena Irimia , Maryam Dayeri
Registered students (2nd project): Yana Tarasova , Esteban López Tavera , Аlena Famina , Maryam Dayeri, Sergey Zvonarev
Accepted students: Irimia Ramona-Elena , Sergey Zvonarev
Final presentation: Irimia Ramona-Elena , Sergey Zvonarev - Recombinant protein production of a restriction-modification enzyme Type I in a fermenter

Project´s aim:

Cultivation of E. coli containing the expression system for suitable protein in a fermentor and for comparison in Erlen-Mayer flasks.
Real time observation and optimization of cultivation conditions by regulation of nutrition and oxygen.
Step examination of protein expression during cultivation.
Purification of proteins obtained from the cultivations.

Results: Comparison of protein yield from classical batch culture and from fermentor cultivation.

Short annotation (EN):

Restriction modification systems type I are multifunctional complexes, which protect bacterial cells against foreign DNA. Large amount high quality protein sample are essential for structural and functional chanracterization of these protein systems. Thus, a high ratio of expressed recombinant protein level in relation to basal protein expression is demanded. Cultivation in a fermentor allows us to set up cultivation conditions for high protein expression by controling air, stirring and the nutrition input. This project gives will give the participant an overview of microbial cultivation techniques and semi-prepapative chromatography.

4. Project : Molecular mechanism of bacterial Type I restriction-modification enzymes.

Registered students (1st project): Maryam Dayeri , Аlena Famina , Sandeep C , Hanna Krylova , Mihail Shaprio
Registered students (2nd project): Ramona-Elena Irimia , Eduardo Cárdenas , Ekaterina Nikitenko , Maryam Dayeri , Sergey Zvonarev
Accepted students: Hanna Krylova
Final presentation: Hanna Krylova - Molecular mechanism of bacterial Type I restriction-modification enzymes

Project´s aim:

Biochemical and biophysical study of the various enzymatic activities of bacterial type I restriction-modification enzymes. Detailed study of reaction kinetics, and inter and intra molecular interactions.

Results: Comparison of enzymatic activities of wild type enzyme and protein containing single-point mutations, and functional assignment of the mutants.

Short annotation (EN):

Restriction-modification systems are multifunctional complexes and act as a “primary immunity system” in the protection mechanisms of prokaryotic cells from foreign, invading DNA. Type I restriction enzymes are ATP-dependent DNA translocases, and their mechanism of action is not fully understood despite the available biochemical and structural information. Our project is focused on the motor subunits (HsdR) that are responsible for DNA-translocation and DNA-cleavage. The project shall contribute to the understanding of the molecular mechanism of translocation and cleavage while studying reaction kinetics and inter and intra molecular interactions of restriction enzymes Type I as complexes. Participating students will get in touch with biochemical and molecular biology methods, such as site direct mutagenesis, enzyme assays, protein expression and purification, limited proteolysis, visualization methods, as well as with two biophysical methods, surface plasmon resonance (SPR), and isothermal titration calorimetry (ITC).

5. Project : DNA-binding proteins. Purification and Properties

Registered students (1st project): Yana Tarasova , Ekaterina Nikitenko, Anastasia Zharskaya , Sergey Zvonarev
Registered students (2nd project): Jagat Rathod , Ekaterina Nikitenko , Sopio Melikishvili , Attila Surányi , Marianna Sohová , Beáta Kaľatová
Accepted students: Beáta Kaľatová
Final presentation: Beáta Kaľatová - How point mutations in the motor subunit influence on activities of EcoR124I restriction-modification complex?

Project´s aim:

During the project, students will be able to get practical experience in production, purification and analysis of biochemical/structural properties of DNA-binding proteins.

Results:

DNA-binding protein of interest will be expressed, purified and its DNA-binding properties will be analyzed.

Short annotation (EN):

Activities will include standard biochemical techniques including protein expression, purification and analysis of proteins properties as they complex with DNA.

6. Project : Novel genetically encoded sensors of cellular processes

Registered students (1st project): Marina Parr , Bahareh Shakiba , Eduardo Cárdenas , Ekaterina Nikitenko , Esteban López Tavera
Registered students (2nd project): Ekaterina Nikitenko , Anastasia Zharskaya , Adela Vodakova
Accepted students: Nikitenko Ekaterina

Project’s aim:

The aim of the project is to create usable genetically encoded fluorescent sensors of voltage and intracellular calcium concentration for monitoring of neuronal activity. The aim will be achieved by optimization of existing sensors for utilization with two-photon polarization microscopy, as well as by creation of novel genetically encoded sensors. The newly developed sensors will improve our understanding of molecular processes in living cells and organisms.

Short annotation (EN):

Students will take part in development of genetically encoded sensors of intracellular calcium concentration and cell membrane voltage. Students will create DNA constructs encoding the sensors using methods of molecular biology (PCR, molecular cloning), and introduce these constructs into living mammalian cells by transient transfection. Functional activity of the constructs will be determined using methods of one-photon wide field microscopy, and two-photon polarization microscopy. Students will carry out data analysis in ImageJ and Matlab.

7. Project : Development of two-photon polarization microscopy into a protein structure tool

Registered students (1st project): Natalia Marukovich , Sopio Melikishvili , Adela Vodakova
Registered students (2nd project): Alina Pranovich, Štěpán Timr, Josef Melcr
Accepted students: Josef Melcr , Adéla Vodáková
Final presentation: Adéla Vodáková - Development of two-photon polarization microscopy into a protein structure tool
Final presentation: Josef Melcr - Development of two-photon polarization microscopy into a protein structure tool

Project’s aim:

The aim of the project is to develop the capabilities of two-photon polarization microscopy (Lazar & al., Nature Methods 2011) to yield insights into structure of membrane proteins, directly in living cells and organisms.

Short annotation (EN):

The project activities will bridge cell biology, molecular biophysics and structural biology. Students will obtain information about optical properties of fluorescent proteins and synthetic dyes, and apply this information towards determining structural properties of model membrane proteins. Students will gain experience with a diverse range of skills (crystallography of fluorescent proteins, methods of molecular biology and mammalian cell culture, advanced optical and microscopy techniques, mathematical modeling, molecular dynamics simulations, and structural biology). The project is highly multidisciplinary, not driven by a particular technique, but rather by a major scientific question: what is the structure of membrane proteins in living cells? To make the project both enjoyable and stimulating, students will be provided both with guidance and with independence (matching their abilities).

8. Project : Superresolution two-photon fluorescence microscopy using photoswitchable fluorescent proteins

Registered students (1st project): Alina Pranovich, Štěpán Timr , Marianna Sohová , Beáta Kaľatová , Josef Melcr
Registered students (2nd project): Natalia Marukovich, Marina Parr
Accepted students: Marianna Sohová
Final presentation: Marianna Sohová - Superresolution two-photon fluorescence microscopy using photoswitchable fluorescent proteins

Project’s aim:

The main goal of the project is to combine the method of two-photon polarization microscopy that we recently developed (J.Lazar & al., Nature Methods, 2011) with microscopy techniques that allow breaking the diffraction limit on resolution of optical microscopes. The resulting microscope and molecular probes should allow detailed observations of molecular processes taking place in living cells and, ultimately, in a living brain.

Short annotation (EN):

The project is highly multidisciplinary, and will be accomplished through a combination of techniques of molecular biology, cell biology, optics, optical engineering, and mathematical modeling. Using techniques of molecular biology we will prepare DNA constructs based on photoswitchable fluorescent proteins. These constructs will then be introduced into cultured mammalian cells, and the fluorescent cells will then be used to carry out superresolution observations of molecular processes. In order to accomplish these observations, we will have to modify our highly versatile, one-of-a-kind optical microscope. Data acquired by superresolution imaging will be processed by software, and analyzed by using mathematical modeling. Participating students will be exposed to many cutting-edge techniques, but most importantly, to world class science focused not on a particular technique, but on answering a major scientific question: how does the brain work? To make the experience both enjoyable and stimulating, students will be provided both with guidance and with independence matching their abilities.

9. Project : Structure and dynamics of biological relevant molecules in pure and aqueous solutions of ionic liquids

Registered students (1st project): Dzmitry Shemel, Béla Fiser
Registered students (2nd project): Hanna Krylova , Mihail Shaprio , Sergey Golenchenko , János Szórád
Accepted students: Mihail Shapira , Béla Fiser
Final presentation: Mihail Shapira , Béla Fiser - Interaction of soil organic matter with biomolecules a computational study

Project´s aim: This project aims to demonstrate and reveal solvation and dynamics of biomolecules in non-aqueous media such pure and aqueous solutions of ionic liquids in order to understand and predict the effect of such solutions to biological relevant molecules by theoretical and computational methods such molecular dynamics simulations.

Results: Results of this study will show how non-native environment of biomolecules can influence of the structure of biomolecules which help us to develop new environment for stabilization of biomolecules.

Short annotation (EN):

Solvation in non-aqueous solutions of biomolecules such as peptides, proteins and enzymes and charged molecules in general plays an important role in many areas of chemistry such as physical chemistry, solution chemistry, electrochemistry, biological chemistry and solvation process is a key issue in applied chemistry. A deeper understanding of the solvation phenomenon in complex systems, such as biologically relevant molecules in non-aqueous solutions is crucial for many applications of non-aqueous biocatalysis. The usage of organic solvent and ionic liquids in many cases can improve the applications of enzymes with decreasing the association in aqueous solutions by increasing the solubility of hydrophobic substrates. In this project, we would like to study of small biomolecules such as small hydrophobic and hydrophilic peptides and small proteins such as lysozyme in the presence of different solutions of hydrophilic and hydrophobic ionic liquids in order to reveal their solvation structure, dynamics and stability to relate them to their activity in non-aqueous solutions. As our methodology is based on molecular dynamics (MD) simulations we will analyzing the molecular dynamics simulation we will reveal the influence of none-aqueous media on the structural changes of such biomioleculs in terms of thermal stability and influence of non-aqueous media on the structure of the active sites of enzymes. As the properties of ionic liquids can be tunned by using different cations and anions thus enzymatic activity and structural stability can be studied by tunning the hydrophilicity or hyrophobicity of ionic liquids. This project aims to demonstrate and reveal solvatio