P R O J E C T S
1. Computational modeling of Human 3’-phosphoadenosine 5’-phosphosulfate Synthase PAPSS: Gene sequence variation among human populations and its consequences on protein structure/functions.
By computational modeling gain a better understanding of the structural and functional consequences of DNA mutations in Human 3’-phosphoadenosine 5’-phosphosulfate Synthase PAPSS among various human populations. The results will greatly contribute to the molecular understanding of diseases caused by deficiency in intracellular sulfate as is autosomal recessive disorder osteochondrodysplasiae.
Understanding how the three dimensional structure of PAPSS determines the enzyme function? Studying the roles of specific amino acid residues/overall structures, on the dynamics of the enzyme in aqueous solution and the related quaternary arrangements of the enzyme. To predict/describe enzymatic reactions in three-dimensional space and to explore the reaction coordinate through the lens of molecular dynamics simulations.
Sulfonation is important for life. Upon sulfonation molecules become more polar and more soluble in aqueous solutions. In Golgi bodies sulfuryl groups are added to proteins and carbohydrates that are later exported from the cell, including the heavily glycosylated proteoglycans which are the major components of the mammalian extracellular matrix. Thus, a deficiency in the intracellular sulfate has severe clinical consequences since under sulfated proteoglycan leads to autosomal recessive disorder osteochondrodysplasia. Osteochondrodysplasia is characterized by short limb stature, cleft palate, generalized dysplasia of joints, and hitchhiker thumb that are typical physical characteristics of these skeletal disorders. Since inorganic sulfate is biochemically inert and cannot be used by the cell directly, how is the active sulfuryl donor made? Activated sulfur is 3’-phosphoadenosine 5’-phosphosulfate (PAPS), a sulfur nucleotide. PAPS is the universal sulfuryl donor of the cell. In mammals 3’-phosphoadenosine 5’-phosphosulfate Synthase (PAPSS), using ATP, converts biochemically inert inorganic sulfate to the metabolically active PAPS . It is a bi-functional enzyme and catalyzes the formation of PAPS in two sequential steps. Since functions of proteins are determined by their structure, corresponding gene mutations could cause altered protein structure/function. Since different human isoforms of PAPSS (hPAPSS1, hPAPSS2a and hPAPSS2b) are exclusively responsible for the synthesis of PAPS, mutations in the PAPSS genes could cause severe disease states. Understanding the global structure/function outcomes of the enzyme due to DNA sequence variations among various human populations involves a detailed description of the protein dynamics. Point mutations, aside from forming potential direct interactions with the substrate, might also redistribute the protein conformational ensembles. Since crystal structures of PAPSS1 with various ligands and combinations have been resolved by X-ray diffraction to a resolution of up to 1.75 Å (PDBs: 1x6v, 2ofw, 1xjq, 1xnj) and for PAPSS2 a crystal structure of the kinase domain is available with 2.5Å resolution (PDB: 2ax4), we can apply a molecular dynamics approach to hPAPSS and perform comparative bioinformatics and simulation studies. Understanding the structural and functional consequences of DNA mutations among various human populations will greatly contribute to the molecular understanding of the above described diseases and will allow the designing of better drugs targeted towards specific population groups/individuals.
2. Crystallographic studies of the HsdM subunit in type I restriction modification system EcoR124I
The project spans around exploring the methods for overexpression of EcoR124I HsdM subunit and various purification steps involved, preparation of protein samples for obtaining crystals, optimizing crystallization conditions and structure determination using x-ray crystallography.
Obtaining good-sized, diffraction-quality crystals, collect diffraction data for solving atomic-scale structure of the EcoR124I HsdM subunit.
The type IC EcoR124I systems comprises of HsdS and HsdM subunits that form the core methyltransferase (MTase) complex. Besides, the target DNA sequence is recognized by HsdS (specificity), restriction carried out by HsdR which shows ATPase activity which is responsible for DNA translocations as well (restriction/cleavage), methylation of the adenosine residues is carried out by HsdM (modification). This complex behaves both as an endonuclease and as a MTase, depending on the methylation status of adenine residues in the target sequence, three enzyme subunits (1S and 2M) either act together as a typical MTase or recruit a pair of HsdR subunits that initiate translocation of DNA through the enzyme and eventually cleave non-specifically at apparently random sites in an Adenosine-5′-triphosphate (ATP)-dependent manner.
Three-dimensional structure of a protein molecule is quintessential for studying its assembly, association with other molecules and biological functions. X-ray crystallography is a major technique to solve the 3D structures of biomacromolecules. However the challenge is to obtain high-resolution diffraction data for which a stable, good-sized single crystal is necessary. The art of obtaining crystals of adequate size and quality to permit accurate data collection depends largely on the purity of the protein sample (>90-95%) The key to obtain ordered crystals that diffract x-rays is the gradual precipitation of protein under suitable conditions over a period of few days to several months in a trial-and-error manner. Vapor diffusion method remains the most common and preferred, and the crystal growth is preceded by protein precipitation and nucleation stages. The ideal size for a crystal to be used for a diffraction study varies between few hundred and 20 microns. The optimal conditions rely on various factors such as the concentration of the salts, organic compounds, additives etc. in the precipitant solution and by varying the protein concentrations. Once the crystals are obtained, a detailed three-dimensional structure of a protein and the complexes can be constructed based on the pattern of diffraction obtained.
3. Modeling interactions in biomolecules using methods of quantum and molecular mechanics
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.
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.
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
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.
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.
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
The aim of the project is to determine whether cholesterol in plasma membrane affects conformation and functional activity of heterotrimeric G proteins.
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
To develop a fluorescent protein suitable for observing electrical signals in neurons.
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
To develop two-photon polarization microscopy into a novel quantitative technique of structural biology.
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
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.
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.
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. Biochemical analysis of membrane proteins in yeast
Mariia I. Borbuliak
Analysis of the oligomeric state of yeast K+-translocation proteins (Trk1 and Trk2) using biochemical methods (SDS-PAGE, Western blot).
TRK proteins are cation translocating proteins that allows baker’s yeast (Saccharomyces. cerevisiae , S.c) to survive and grow under different environmental [K+] ranging from a few µM to hundreds of mM and maintaining internal K+ concentration relatively constant. Intracellular K+ plays an important role in essential cellular processes, such as osmo- regulation, protein synthesis and regulation of enzyme activities. It is also required for negative charge compensation. In S.c there are two specific K+ translocation systems: Trk1 and Trk2. Both are large membrane proteins made out of 1235 (Trk1) and 889 (Trk2) amino acid residues, respectively. It is presumed that they exist in the plasma membrane as (homo- or hetero) dimers or tetramers. In this project we will work on establishing methods to analyse the “multimerity” of Trk1 and Trk2. Main aim is the standardisation of a protocol for the isolation of intact Trk1 and Trk2 from yeast expressing Trk1- and Trk2-green fluorescent protein (GFP) fusion constructs. This will include cell membrane isolation using different methods (e.g. disruption with glass beads or “French Press”). Protein concentration will be determined using Bradford’s and other methods. Analysis of isolated fractions will be performed using different types of SDS-PAGE gels (Tris-Glycine, Tris-Tricine, Tris-Acetate). The integrity of Trk(1,2)/GFP will be examined by western blot followed by immunostaining of the blots using Anti-GFP antibodies. Other methods used within this project will include microbiological techniques like maintenance and growth of yeast cells and fluorescence microscopy to examine the cellular localisation of Trk/GFP fusion proteins.