Projects

1. Crystallization of proteins with Ionic Liquids

Registration for this project closed
Project leader: M. Sc., Ekaterina Tutubalina, tutubalinakate@mail.ru
Associate Professor: Ivana Kuta Smatanova, Ph.D., ivanaks@seznam.cz
Registered students: Mohamed, Hussien Sallama, Vamush, Sibanbaeva, Markevych, Mohamed, Baba, Zvonarev, Shchur, Ponomareva, Roeselova

Chosen student:

 Mihail Shapira

Project´s aim:
During the Summer School 2014 students will study basic information about protein, its parameters, properties and structure and how do they form crystals. Also they will know how to crystallize protein samples using several standard methods and modern crystallographic techniques; find optimal condition due to obtain “nice’ crystals of proteins trying to use all crystallization methods and broad spectrum of precipitant solutions.
After getting basic knowledge, selected enzymes (model proteins such as lysozyme, thaumatin, glucose isomerase) will be crystallized with ionic liquids as additives in various crystallization plates (Hampton Research – CA, USA, MDL – UK, Emerald BioStructures, WA, USA) for hanging or sitting drops and in capillary tubes (Triana Sci & Tech, Spain) at room temperature and at 4 deg. Conditions that yield crystals of any kind will be repeated once again but without ILs to compare influence of ILs on crystallization process (quality of crystals, their shape and size, etc).
Results:
Determine ILs influence on protein crystallization, study dependence of size and properties of particular IL on protein crystallization.
Short annotation:
The fact that many enzymatic catalytic reactions take place in aqueous solutions, some of them can also take place in non-aqueous solutions of organic solvents or ionic liquids (ILs), can open a new opportunity for scientists to work with enzymes, which are unstable in aqueous solutions. Ionic liquids (ILs) are organic salts composed of separate cations and anions, which are liquid at room temperature. They often have negligible vapor pressure, high thermal stability, but their key feature is their ‘tuneable’ nature. Indeed, they can be designed to have specific physical and chemical qualities by acting on their alkyl chains (i.e. modifying the length, the presence of hydrophobic groups, etc.) or the anion (i.e. varying the degree of the charge localization, its hydrogen bonding ability, etc.). The use of ILs as solvents for biological applications is currently receiving considerable attention, and ILs have been used successfully as refolding additives for protein renaturing studies, increased protein shelf life, enhanced thermal stability, and as additives which increase enzymatic reaction rates. We suggest that using of ILs will increase the frequency of obtaining the crystals and also improve their quality.


2. Crystallization of haloalkane dehalogenase LinB mutants

Registration for this project closed
Project leader: Iuliia Iermak, MSc, julia.ermak90@gmail.com
Registered students: Mashaparne, Markevych, Mohamed, Baba, Nahornaya, Nahornaya, Roeselova
Chosen student:

Alzbeta Roeselova
Mykyta Markevych

Project´s aim:
Haloalkane dehalogenase LinB isolated from a bacterium Sphingobium japonicum UT26 has relatively broad substrate specificity and can be potentially used for biosensing and biodegradation of environmental pollutants. During the project the student will study various techniques of proteins crystallization such as sitting drop vapour diffusion, hanging drop vapour diffusion and microseeding. These techniques will be then applied for crystallization of LinB mutants.
Results:
Results of this project will show features of crystallization behaviour of LinB mutants and obtained crystals can be used for structural characterization of these enzymes.
Short annotation:
LinB is a microbial enzyme that belongs to the family of haloalkane dehalogenases. Substrates for LinB are monochloroalkanes, dichloroalkanes, bromoalkanes and chlorinated aliphatic alcohols. These halogenated organic compounds represent one of the largest groups of environmental pollutants. LinB isolated from a soil bacterium Sphingobium japonicum UT26 can be applied in biodegradation of environmental pollutants. Different LinB variants were constructed for studying the effect of mutations on enzyme’s functionality and it is highly important to have structural information about each mutant. Nowadays one of the main techniques that are widely used for structural characterization of macromolecules is X-ray diffraction analysis of macromolecular crystals. In this case the bottleneck in structure determination is obtaining good quality protein crystals. Within this project the student will learn different crystallization techniques and their application for crystallization of haloalkane dehalogenase LinB.


3. Expression, purification, restriction and ATPase activities in mutated subunit HsdR from EcoR124I restriction-modification complex

Registration for this project closed

Project leader: Mgr. Katsiaryna Shamayeva, shamayeva@nh.cas.cz
Registered students:Hussien Sallama, ragheb, Emam, Fiser, Varatnitskaya, Maskalenka, Nahornaya, Nahornaya, Popa, Shchur, Ponomareva
Chosen student:

Gabriela Fiser

Project´s aim:
During the project, students will be able to get practical experience in production, purification and analysis of biochemical properties of proteins.
Results:
HsdR mutated subunits from EcoR124I from E.coli will be expressed, purified and its DNA-binding properties will be analyzed.
Short annotation:
Restriction-modification enzymes protect bacteria against foreign DNA. EcoR124I is type I restriction-modification complex with 5 subunits: HsdS subunit is responsible for DNA specificity, two HsdM subunits is responsible for DNA methylation and the biggest part of whole complex is HsdR subunit is responsible for DNA translocation and cleavage. Two HsdM and HsdS subunits perform independent complex MTase. HsdR subunit includes four domains: endonuclease, two Rec-A-like helicase domains and helical domain. Our work will be focused of HsdR subunit with different point mutations in different domains. Participant students should find expression conditions for mutated HsdR, purified mutants and analyzed it using biochemical methods.


4. Modeling interactions in biomolecules using methods of quantum and molecular mechanics

Registration for this project closed

Project leader: David Řeha, Ph.D., reha@nh.cas.cz
Registered students: Jessiebiz, Strumillo, Korneeva, Kuzmitskaya, Sláma, Marzec, Chubarova, kapustsin, Zhao, Šimko, Stančiaková, Kviatko, Kozlenkova, Hernández-Oropeza
Chosen student:

Pavel Kviatko
Marta Strumillo

Yani Zhao

Project´s aim:
The study of interactions between proteins and several ligands (drugs) and other related bimolecular processes by means of various computational methods, particularly quantum mechanics (QM), hybrid QM/MM methods, molecular dynamics (MD) simulations and molecular docking.
Results:
The position of the various ligands (drug agents) within the protein will be calculated by the methods of molecular docking. The accurate binding energy of the ligands to the protein will be calculated using QM/MM calculations. The dynamic properties of the protein complexes will be then investigated using MD simulations. The results of computational modeling will be compared with experimental results.
Short annotation:
Computational methods are important tools in study of biomolecules including their interactions with other molecules (pharmaceutical drugs) and bimolecular processes. Within our project we would like to focus on very accurate description of the active site of the proteins and their interactions with ligands, substrates or protein co-factors. Such a level of accuracy can be only achieved by methods of quantum mechanics (QM). Since QM calculations are computationally very demanding and the description of the large biomolecules by purely QM methods is very limiting, hybrid QM/MM methods would be employed. QM/MM methods combine QM for calculations of active site and method of molecular mechanics (MM) for the calculation of the rest of the system. We would like to apply QM/MM methods for calculation of the binding energy of various ligands in proteins (NADH and FMN in flavoproteins, drugs in plasma proteins, etc). We would also like to employ methods of polarized molecular docking (based on QM/MM) in order to predict the geometry of various ligands in binding sites of the proteins.


5. Theoretical investigation of the interactions of hydrated ionic liquids with membranes for bio-applications and drug delivery

Registration for this project closed
Project leader: M.Sc. Babak Minofar, PhD, minofar@nh.cas.cz, babakminoofar@gmail.com
Registered students: ragheb, Koshcheyev, Marzec, Chubarova, kapustsin, Šimko, Stančiaková, Voronina, Shapira, Vileishikova, Hernández-Oropeza, Ferencová
Chosen student:

Stanislav Šimko
Katarína Stančiaková

Project´s aim:
The objective of this project is to study theoretically the interaction of aqueous solutions of ionic liquids with biologically related compounds in order to understand their roles in possible bio-applications such as drug delivery, protein folding and protein crystallization.
Results:
The results of the project will show us how protic and aprotic ionic liquids for example biocompatible ionic liquids such as choline based or alkyl ammonium ionic liquids with different anions as can interact with biomolecules in order to clarify their possible applications in future for drug deliver.
Short annotation:
Understanding the mechanism of ion associated pharmaceutically active ionic liquids with membranes can bring crucial information for the transport process of pharmaceutical active salts across the membrane and reaching the active site.1 Transport process of compound such as ion or substrate through biologically related membranes or nanopores is very important in research as they have very important applications in biological systems. The translocation process of substrates such as antibiotics, DNA,and peptides through nanopores have been studied using electrophysiology.2-4
Ionic liquids (ILs) are organic salts which have liquid property at room temperature with many interesting and characteristic properties such as low vapor pressure, low volatility and stability which make them to be known as environment-friendly or green solvents. These properties of ILs make them to be used in many biological and chemical reactions therefore they are used in many research and industrial applications from chemical industries to pharmaceuticals and food industries.3,4In order to use ILs for pharmaceuticals as potential future drugs understanding their mechanism of toxicity the physical and biological interactions between cells must be carefully studied both experimentally and theoretically. In this study the interactions of biological membranes with hydrated ILs will be studied by molecular dynamics (MD) simulations in order to reveal the possible perturbation of membrane surface and penetration to the lipid bilayers by using ILs with different cations and ionic with wide verity of hydrophobicity character.
Both protic and aprotic ILs such as choline or alkyl ammonium based ILs with different anions as biocompatible and biodegradable materials will be used for simulations to study perturbation of membrane surface and possible penetration of them through model membrane bilayers such as dipalmitoylphosphatidyl choline (DPPC) or (2R)-3-hexadecanoyloxy-2- [(Z)-octadec-9-enoyl]oxypropyl]2-(trimethylazaniumyl)ethyl phosphate (POPC) as models of the biological membranes.


6. Molecular mechanisms of G protein signaling investigated by two-photon polarization microscopy

Registration for this project closed
Project leader: A.Bondar/J.Lazar, bondar@nh.cas.cz , lazar@nh.cas.cz
Registered students: Badwy , Mystek, Kuzmitskaya, Krutyholova, Kuryltso, Sawa, Havarouski, Palianina, Palianina, Sibanbaeva, Ralovets, Barashkava, Maskalenka, Kozlenkova, Takahashi
Chosen student:

Katsiaryna Maskalenka 
Kajetan Sawa

Project´s aim:
The aim of the project is to determine whether cholesterol in plasma membrane affects conformation and functional activity of heterotrimeric G proteins.
Short annotation:
G proteins and G protein-coupled receptors are key players of cell signaling and intercellular communication. They detect and transduce signals from a multitude of physical and chemical stimuli, including hormones, neurotransmitters, odorants, light, flavors, etc. We are interested in molecular mechanisms of signal transduction through various G-proteins. It has been proposed that certain types of G proteins localize to cholesterol-enriched membrane compartments, such as lipid rafts or caveolae. Cholesterol of these compartments is thought to affect the G protein conformation and regulate the G protein functional activity. We will determine the role of cholesterol in G protein signal transduction by studying fluorescently labeled G proteins in intact and cholesterol-depleted cells using the technique of two-photon polarization microscopy developed in our lab (Lazar et al Nature Methods 2011). During the project students will use a number of different experimental techniques, including methods of molecular biology, cell biology, microscopy and biochemistry. Students will also perform quantitative image analysis and take part in modeling of experimental systems using the methods of molecular dynamics. The project is aimed to provide the students with the opportunity to take part in state-of-the-art scientific research using cutting-edge experimental methods.


7. Development of fluorescent proteins sensitive to cell membrane voltage

Registration for this project closed
Project leader: J.Lazar, lazar@nh.cas.cz
Registered students: Badwy , Emam, Strumillo, Koshcheyev, Mystek, Baster, Żak, Krutyholova, Kuryltso, Czuchnowski, Sawa, Voronina, Chernikovich, Reukova, Kulik, Kviatko, Ferencová, Novotná , Takahashi
Chosen student:

Ivan Kulik

Project´s aim:
To develop a fluorescent protein suitable for observing electrical signals in neurons.
Short annotation:
The brain is an electric organ. In order to understand how the brain works, we need to be able to visualize electrical signals in neurons. Being able to see, using a microscope, the electrical signals traveling through the brain would revolutionize neuroscience. Although significant progress in this direction has been made in recent years, there is still much room for improvements in genetically encoded optical probes of cell membrane voltage. Two-photon polarization microscopy, an advanced optical microscopy technique developed in our laboratory (Lazar & al., Nature Methods 2011) offers new ways to observe changes in cell membrane voltage. The goal of the project is to investigate the ability of two-photon polarization microscopy to visualize changes in cell membrane voltage, using both existing and novel voltage sensitive fluorescent proteins. During the project, students may use a wide range of techniques, including methods of molecular biology, cell biology, single cell electrophysiology, advanced microscopy and biological image analysis. The research is conducted in collaboration with laboratories at Yale University. The project is aimed to provide students with the opportunity to take part in state-of-the-art scientific research using cutting-edge experimental methods.


8. Development of optical microscopy into a structural biology technique

Registration for this project closed
Project leader: A.Kevorkova/J.Lazar, kevorkova@nh.cas.cz , lazar@nh.cas.cz
Registered students: Baster, Sláma, Żak, Czuchnowski, Reukova, Fiser, Ralovets, Kulik, Vileishikova, Barashkava
Chosen student:

Elena Vileishikova
Jakub Czuchnowski
Maryia Barashkava

Project´s aim:
To develop two-photon polarization microscopy into a novel quantitative technique of structural biology.
Short annotation:
Most techniques of structural biology, such as X-ray crystallography or NMR spectroscopy, typically provide information about structure of proteins as they exist outside of cells. In contrast, techniques of optical microscopy (such as two-photon polarization microscopy, Lazar & al., Nature Methods 2011) have the potential to yield information about structure of proteins directly in living cells. The goal of the project is to contribute towards development of optical microscopy into a technique capable of providing detailed, quantitative information on structure of proteins (in particular membrane proteins) in living cells. During the project, students will work with fluorescent dyes and fluorescent proteins, using both in vitro and living systems, and perform advanced microscopy experiments (non-linear optical microscopy, superresolution microscopy) . They may use a wide range of techniques, including methods of molecular biology, biochemistry, cell biology, molecular dynamics simulations, advanced microscopy and biological image analysis. The project is part of an ongoing collaboration with the European Synchrotron Radiation Facillity in Grenoble, and is aimed to provide the students with the opportunity to take part in state-of-the-art scientific research using cutting-edge experimental methods.


9. Monitoring intracellular pH changes of yeast cells

Registration for this project closed
Project leader: Jost Ludwig, jost.ludwig@uni-bonn.de
Registered students: Markéta Novotná , Sergey Zvonarev , Madalina Oana Popa
Chosen student:

Daria Polyanina
Madalina Oana Popa

Project´s aim:
Analysis of intracellular pH changes of yeast (Saccharomyces cerevisiae) cells upon changes in extracellular pH and external K+ concentration. In the project we’ll generate different yeast strains (carrying mutations in K+ translocation system genes) producing the genetically encoded pH sensor pHluorin. These strains will be verified by fluorescence microscopy. Eventually time resolved measurements of intracellular pH will be carried out using a fluorescence microplate reader. Mainly the response of intracellular pH upon changes of external pH and external K+ concentration will be analysed.
Results:
Generation of transgenic yeast strains, comparison of intracellular pH changes dependent on (i) extracellular pH and (ii) and external K+ concentration. Influence of K+-translocation systems.
Short annotation:
The intracellular H+ ion concentration (usually expressed as pH) is an important determinant of the ability of cells to perform their tasks. Therefore, cells usually try to keep their intracellular pH constant in order to provide optimal conditions for enzyme activity. However, changes in the extracellular pH also can lead to changes in intracellular pH that have to be compensated by the cell. While this problem is almost non-existing in cells living in a more or less stable environment (like most mammalian cells), yeast cells have to adapt to strongly changing environments. Since H+ is charged, all translocations of H+ ions are accompanied by a change in the membrane potential that in turn also has to be compensated. This is (one of) the reason(s) why H+ homeostasis is strongly connected to K+ (the most abundant intracellular cation) homeostasis. Measurements of intracellular pH can rather easily carried out using cells expressing the gene encoding for pHluorin, a GFP (green fluorescent protein) variant that changes its fluorescence properties depending on pH. During the summer school we’ll generate yeast strains (differing in the presence of K+ translocation proteins) expressing the pHluorin gene, verify them and use these strains to monitor (highly time resolved) changes of intracellular pH upon changes in extracellular pH and the presence of K+.
The methods used will be (i) molecular biology (plasmid preparation, analysis by restriction digestion, PCR), (ii) fluorescence analysis (microscopy and quantification of fluorescence using a microplate reader) microscopy and (iii) biophysics (mathematics) for data evaluation.