P R O J E C T S
1. Expression, purification and crystallization of photosynthetic protein PsbR using different vectors and crystallization techniques.
Project leader: Mgr. Jiří Heller, hellej01@prf.jcu.cz
Chosen students: Artem Dubovetskyi, Valeria Kopats
Project´s aim:
During the project the student will be introduced into the field of molecular biology methods such as protein overexpression, purification using AKTA and others like: CD, fluorescence and DLS measuring prior to prepare protein sample suitable for crystallization. Basic crystallization techniques will follow and the student will learn to distinguish different structures which will occur in the drop.
Results:
Obtain crystals of PsbR protein.
Short annotation:
The PsbR protein was firstly mentioned in 1986. That time it was firstly isolated from PS II of thylakoids of higher plants as a 10 kDa polypeptide. Unluckily, because of its high unstability, scientists even did not managed to sequence it to elucidate its nucleotide sequence. On the contrary they were capable to discover that it did not contain any of the substances with absorption of light or metallic cofactors. In addition the protein exhibited some hydrophobic properties. A supposition was made that this 10 kDa protein could act as a binding site for the extrinsic 23 kDa (PsbP) protein in the thylakoid membrane (Ljunberg et al., 1986). Just three years later in the paper on wheat was written that this 10 kDa peptide connected with the oxygen-evolving complex contained a non-cleavable putative C-terminal domain (Weber et al., 1989). Now we know that it is a part of OEC of photosystem II, but unfortunately its exact function and position remains almost 30 years after its discovery unknown, we even do not know with wich other protein it interacts and only some models were made.
2. Crystallization of glyceraldehyde dehydrogenase or how to overcome the main bottleneck of macromolecular crystallography
Project leader: Iuliia Iermak, MSc, julia.ermak90@gmail.com
Chosen students: Bianka Kőhegyi, David Novak
Project´s aim:
During the project students will go through all steps of protein structure determination from obtaining a good-quality crystal to the structure refinement of glyceraldehyde dehydrogenase from Thermoplasma acidophilum (TaAlDH). Crystallization part will include optimization of obtained crystallization conditions by variation of protein and precipitant concentrations and pH and using microseeding procedure. In the second part students will study how to work with software for diffraction data processing and structure refinement.
Results:
Diffraction-quality crystals of TaAlDH and improvements in its crystal structure should be obtained as a result.
Short annotation:
The glyceraldehyde dehydrogenase from Thermoplasma acidophilum is a part of cell-free system for production of isobutanol and ethanol from glucose. It participates in oxidation of Dglyceraldehyde to D-glycerate in this synthetic pathway. Wild type of TaAlDH has high substrate selectivity and product tolerance but leaves place for optimization so in order to improve enzyme properties various mutants of TaAlDH were constructed using random approach. Still, for further enhancement of the enzyme knowledge of its three-dimensional structure will be useful. Nowadays one of the main techniques that are widely used for structural characterization of macromolecules is X-ray diffraction analysis of macromolecular crystals. Crystals of TaAlDH wild type and two mutants were already obtained; however, optimization of crystals quality is required. During summer school we will use for this purpose alteration of precipitant cocktail parameters, such as protein concentration, concentration of different components of precipitant solution and pH, followed by microseeding or other optimization procedures if needed. Also students will study processing of already collected diffraction data set from TaAlDH crystals and solving the phase problem, and then they will work on structure refinement of TaAlDH.
3. Modeling interactions in biomolecules using methods of quantum and molecular mechanics
Project leader: David Řeha, Ph.D., reha@nh.cas.cz
Chosen students: Joanna Macnar, Monika Litvinukova, Sara Matić
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.
4. Theoretical investigation of the interactions of hydrated ionic liquids with membranes for bio-applications and drug delivery
Project leader: M.Sc. Babak Minofar, PhD, minofar@nh.cas.cz, babakminoofar@gmail.com
Chosen students: Kadri GÜLEÇ, Saeid Vakilian
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.
5. Molecular mechanisms of G protein signaling investigated by two-photon polarization microscopy
Project leader: A.Bondar/J.Lazar, bondar@nh.cas.cz , lazar@nh.cas.cz
Chosen students: Viktor Navrulin, Alina Ralovets
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.
6. Development of fluorescent proteins sensitive to cell membrane voltage
Project leader: Josef Lazar, PhD., lazar@nh.cas.cz
Chosen students: Barbora Hoffmannova, Marharyta Semenikhina
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.
7. Development of optical microscopy into a structural biology technique
Project leader: Josef Lazar, PhD., lazar@nh.cas.cz
Chosen students: Olga Rybakova, Robin Krystufek
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.
8. Monitoring intracellular pH changes of yeast cells
Project leader: Jost Ludwig, Dr. rer.nat.habil., jost.ludwig@uni-bonn.de
Chosen students: Sabina Chubanova, Katarína Mackova, Katarina Siroka
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.
9. Single-molecule investigation of mechanisms behind epigenetic regulation of insulin gene
Project Leaders: Tomáš Fessl, Ph.D., Dr Łukasz Bujak, fessl@prf.jcu.cz, lbujak@prf.jcu.cz
Chosen students: Ewa Tratkiewicz, Katsiaryna Nikitsenka
Project’s aim:
The goal of this project is to elucidate structural mechanisms behind epigenetic control of insulin gene. In particular we will assess structural impact of methylation on two candidate regulatory sequences. First, G-rich sequence, with propensity to form G-quadruplex with complex folding topology. Second, complementary C-rich sequence, which is known to be in dynamic equilibrium between i-motif and hairpin in near-physiologic region of pH.
Short annotation:
Epigenetics is currently one of the fastest-growing areas of science and has now become a central issue in studies of development and disease. It could be defined as a heritable change in gene expression that does not affect underlying DNA sequence. Epigenetics ultimately regulates gene activity and expression during development and also in response to external stimuli.
We are interested in structural mechanisms behind this phenomena. Here, we will focus on epigenetic regulation of insulin gene. Sequence of insulin-linked polymorphic region (ILPR) has several stretches with propensity to form regulatory elements such as G-quadruplexes, i-motifs and hairpins. If stable, all of these elements can block several RNA polymerases, thus inhibiting transcription.
ILPR G-quadruplex has been shown to directly interact with insulin. It can form two distinct conformers with parallel and antiparallel orientation of strands. These conformers then show profoundly different affinity to bind insulin, thus contributing to regulation of gene transcription. Complementary sequence has propensity to form i-motif and hairpin. Said i-motif is known to be stable under near-physiological conditions.
Main research questions are:
- Does methylation of ILPR G-quadruplex prioritize particular conformer?
- Does methylation stabilize ILPR i-motif under physiological conditions?
During the project students will learn basic biophysical methods commonly used in research of structure and function of nucleic acids. They will use circular dichroism, basic and advanced methods of optical spectroscopy and microscopy. Nevertheless, the main focus will be on the single-molecule methods, Single-Molecule Foerster Resonance Energy Transfer (smFRET) and Fluorescence Correlation Spectroscopy (FCS). Students will also learn how to prepare lipid vesicles for surface-immobilized experiments and how to process data using advanced statistical methods, such as Hidden Markov Modelling (HMM).