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Introduction:

Epac1 is a protein involved in the activation of Rap-proteins. Its correct functioning allows a multitude of cellular processes, including apoptosis and cell adhesion, proliferation and differentiation. Understanding the functioning of Epac1 could have medical implications, so detailed insight into its structure and dynamics is essential.

Epac1 contains two subunits. The C-terminal catalytic domain performs the Rap activation, the N-terminal subunit is regulatory and sterically blocks the Rap-binding function on the catalytic domain to keep it inactive. Activation of Epac1 involves opening up of the regulatory and catalytic domains. This depends on direct binding of cyclic AMP, a messenger molecule of which the intracellular concentration depends on external stimuli like hormones. From biochemical and crystallographic studies the Physiological Chemistry group at the Utrecht Medical Centre developed a model to describe the step-wise activation of Epac1 upon addition of cAMP. To verify this allosteric model, in a collaboration with this group we set out to study the structure and dynamics of the Epac1 regulatory domain by NMR spectroscopy.

Fig. 1. The Epac1 regulatory domain changes its overall structure upon cAMP binding.

Some aspects concerning the influence of cAMP on the Epac1 structure and dynamics have been studied already. Still several things are still quite obscure and need more attention: (i) The NMR structure of Epac1 in the unbound state needs to be improved (Fig. 1). (ii) Epac1 is “superactivated” by some ligands, as indicated by a higher binding affinity and activity. A clear explanation for this phenomenon in terms of protein structure and dynamics is still unknown, but can be tackled by NMR spectroscopy.

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Introduction:

With the presently available amount of genetic information, a lot of attention focuses on systems biology and in particular on biomolecular interactions. Considering the huge number of such interactions, and their often weak and transient nature, conventional experimental methods such as X-ray crystallography and NMR spectroscopy will not be sufficient to gain structural insight into those. A wealth of biochemical and/or biophysical data can however easily be obtained for biomolecular complexes. Next to experimental data, bioinformatic predictions can be made based for example on amino-acid sequence conservation. Combining the predicted and/or experimental information with docking, the process of modeling the 3D structure of a complex from its known constituents, should provide valuable structural information and complement the classical structural methods.

Goals:

The goal of this project is to investigate how already available interface mapping data obtained from mass spectrometry techniques and bioinformatic predictions can help in the modeling of biomolecular complexes. This information will be translated into structural restraints which will be introduced in the data-driven docking approach HADDOCK developed in the NMR research group. The results obtained from data-driven docking should be compared to a purely ab-initio docking approach based only on shape complementarity and energetic consideration.

Methods:

This project involves computational modeling and bioinformatics approaches. It will consists of the following steps.

• Data mining and bioinformatics analysis of the protein sequences to predict and analyze the binding interfaces. This information will be required in order to model the complexes in absence of MS experimental data to provide a reference point. You will use among other interface prediction methods developed in the NMR research group (see http://www.nmr.chem.uu.nl/whiscy)
• Computational modeling of the complexes by docking using the data-driven docking approach HADDOCK developed in the NMR research group (see http://www.nmr.chem.uu.nl/haddock). The work will be performed on linux workstations with access to computational Beowulf clusters.
• Computational modeling of the complexes by ab-initio docking using web-based docking servers from other groups.
• Structural analysis of the resulting models with a variety of software

Planning & practical aspects:

For this project, the students will work in two groups: one group will investigate the use of experimental data and/or bioinformatic predictions for the modelling of complexes by docking while the other group will concentrate on ab-initio modelling (thus without data). The results of the two different approaches should at the end be compared in the light of the available experimental information.

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Scientific background:

The post genome projects, aiming at functional characterization of individual components, and the relationship between them, is providing a deeper understanding in regulatory processes of complex biological systems. Key towards understanding such regulatory networks are the availability of quantitative information on the interaction between the individual components. These interaction networks are scale free, where few proteins participate in many interactions. Only if it is understood why some proteins interact with many proteins while others show a more restrictive interaction pattern, we can begin to predict complex biological systems, where the nature of the interactions determines the outcome of these systems. To be able to quantify protein-protein interactions we have developed a high throughput GST pulldown assay. Previous experiments revealed that this assay can be used to identify novel protein complexes and determine dissociation constants for interacting proteins. In an ongoing effort to validate this highly sensitive method we want to compare the results that can be obtained with this method with a classical method to identify protein complexes: the yeast two hybrid method.

Research proposal:

The group of Mark Timmers (physiological chemistry) have, by using yeast two hybrid assays, determined the interaction of essentially all human E2 ubiquitin conjugating enzymes with the E3 ubiquitin ligase proteins. Using our previously established high throughput cloning method most of these proteins are cloned in suitable vectors. Following protein expression and purification the interaction between these two classes of proteins will be determined using the GST pulldown assay. Results obtained with this method will be compared with the yeast two hybrid results.

Methods:

• PCR
• Cloning
• Protein purification in small scale using robotics
• Large scale protein purification using HPLC
• HTP protein interaction assays
• Two hybrid assays
• Enzyme kinetics
• Biophysical characterisation of proteins using fluorescence techniques

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Scientific background:

Phosphatidylethanolamine-binding protein 1 (pebp1) also known as raf kinase inhibitory protein (RKIP) or hcnppp has been implicated in a wide variety of biological processes. The molecular mechanism underlying these cellular functions probably depends on the ability of this protein to bind to raf-1 kinase resulting in the inhibition of raf and mapk signaling. Importantly loss of this protein is associated with tumor progression and metasasis in various types of cancer. The x-ray structure has been determined for many RKIP family members and the phosphatidylethanolamine binding pocket has been proposed but not experimentally determined. Furthermore the binding-pocket for phosphorylated tyrosine a plausible target for this kinase inhibitory protein has been identified. We have recently found that sidenafyl, the active component of Viagra, binds to this protein and finally also nucleotides have a weak affinity for the raf kinase inhibitor. To elucidate the role of ligand binding to the protein we previously determined binding pockets for some of these ligands. Preliminary data further suggest that allosteric mechanisms might play a role in the ability of the protein to bind to various ligands. Goal of this research proposal is to confirm the allosteric behavior of this protein

Research proposal:

The available three dimensional structures of the RKIP proteins will be analysed to design mutants that either interfere with ligand binding or that would prevent communication between binding sites. These mutations will be introduced in an expression plasmid for the RKIP protein and transformed into an expression host in the presence of 15N. Proteins will be purified using few HPLC purification steps. For these mutant proteins the dissociation constants (Kd) for the various ligands will be determined and the 15N HSQC spectra will be compared with the wild type protein in the presence or absence of one and two ligands. The observed differences in this so-called fingerprint spectra, will provide information about the structural changes in the protein. Since allosteric changes in the protein are thought to be accompanied with correlated conformational changes at a distance, these results together could provide evidence for allostery. The availability of these spectra for both wild type and mutant proteins in combination with the kinetic information from binding experiments could provide direct evidence for correlated conformational changes and therby for allosteric regulation.

Methods:

• Structural analysis
• Site directed mutagenesis
• Protein expression
• Large scale protein purification
• Determination of dissociation constants using radioactive or fluorescence methods
• Basic NMR spectroscopy (1D, 15N HSQC)
• Spectral analysis using Sparky
• NMR titration experiments to identify the interaction surfaces

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Introduction:

Understanding the molecular mechanisms of gene regulation are fundamental themes in biology and – when extending to failing regulation associated with human disease - in medicine. A textbook example for understanding genetic control and allostery is the lac repressor (LacR), a protein from the bacterium E. coli. When LacR is bound to the lac operator, the expression of enzymes involved in lactose metabolism is prevented. When an inducer binds to the LacR, the protein is released from the DNA and the genes can be transcribed. Structures of LacR, the DNA binding domains and their complexes with DNA have been determined by X-ray and NMR methods, and a wealth of biochemical data exists for this system. However, it is still unknown, how the protein adapts its structure upon inducer binding, and how this signal is translated to a change in operator affinity. The X-ray structures of the free and inducer bound repressor in the absence of DNA could not resolve this, because both structures lack electron density for the DNA binding domain. Structural information of the LacR ternary complex is fully lacking and also the effect of the inducer is unclear since the structures of the core are very similar in the free and bound states.

LacR is a dimer of dimers with two DNA binding sites and four inducer binding sites. Each monomer consists of the N-terminal headpiece, the core or inducer binding domain and the C-terminal tetramerization helix. The functional DNA binding unit of the LacR is the dimer and is held together via an extensive interface on the core domain [Fig. 1]. Binding of inducer to the core causes a reorientation of the N-sub domains of the core which changes both the intra- and inter-molecular interactions in the monomer and in the dimer. Therefore, consequence of binding of inducer to the LacR is 1000-fold lower affinity of LacR for DNA. Thus LacR reacts allosterically to changes in its environment [Fig. 2].

Fig. 1. LacR dimer DNA-complex

Fig. 2. Lac-repressor DNA-complex

Research proposal:

Student will be involved in preparation of repressors with specifically and firmly attached lanthanide probes which will allow for the first time to probe the structural and dynamical changes at a detailed atomic level by high-resolution NMR spectroscopy. Via site directed mutagenesis we will prepare and functionally test LacR dimer with two neighbouring Cys to which lanthanide probes can be attached. In the past several mutations of LacR have been described that had a direct effect on the LacR allosteric mechanism. For that reason we will also prepare repressors with additional mutations and compare their characteristics to wt LacR. For a few mutants biophysical characterisation will be performed using fluorescence methods, protein-DNA interaction assays and NMR spectroscopy. By comparing NMR spectra of labeled LacR in all states we will be able to outline the allosteric signal from DNA binding site to inducer binding site and so compliment our understanding of the allosteric mechanism of the LacR.

Methods:

• Structural analysis
• PCR
• Cloning
• Site-directed mutagenesis
• Bacterial expression
• Protein purification using HPLC
• Protein-DNA interaction assays
• Protein tagging
• NMR
• Biophysical characterisation of proteins using fluorescence techniques

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Introduction:

Membrane proteins are an important class of proteins and they play key roles in several signaling processes and often function as channels or receptors for small molecules of which their surrogates act as drugs. However, due to technical complications, membrane proteins have long remained understudied, as is reflected in the low number of high-resolution structures deposited in the protein data bank (PDB). My research focuses on the development and application of NMR for the study of membrane proteins.

Project:

Since plants cannot just walk away or put on a jacket, they need to protect themselves against cold-, drought- and salt-stress. A family of small membrane proteins was recently discovered to play an important role in the protection of plants against these abiotic stresses, and similar proteins are also found in yeasts, nematodes and bacteria. The hypothesis is that these proteins perform their action by depolarizing the membrane potential by mediating a proton-leak. By studying the 3D structure of these proteins we would like understand their mode of action.

Hypothesis: the membrane proteins depolarize the membrane through a proton leak

The project includes the expression and purification of one of the proteins of this family, the screening of sample conditions to stabilize the protein in a membrane-like (i.e. detergent micelle) environment, the recording and analysis of NMR spectra, collecting structural information from these spectra and the determination of a model for the structure based on these data. At each part of the project you as a master student could contribute.

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Introduction:

Self-assembled biomolecules play a crucial role in the context of neurodegenerative diseases such as Alzheimer’s or Parkinson’s disease. In addition, revealing how such conformational states are formed and stabilized may help to understand and mimic biological functioning. Solid-state NMR (ssNMR) has become the premier method to obtain structural insight into these non-crystalline and heterogeneous systems at atomic resolution.

Project:

While ssNMR has made great progress to study amyloidogenic proteins at the atomic scale, it is still poorly understood how protein sequence and preparation conditions influence the formation of macromolecular networks finally leading to amyloid fibrils. For example, changes in peptide sequence, concentration, solvent, pH and temperature or the presence of molecular seeds have been shown to impact the formation of self-assembled peptide networks. In this project, molecular networks of a series of short peptides shall be made and studied by ssNMR and other techniques under a variety of macroscopic conditions.

Strategy: Produce self-assembled peptides and study the structure at atomic resolution using solid-state NMR

As a master student you will be involved in the preparation of peptide assemblies for an available set of peptide constructs under a variety of experimental conditions and in the recording and analysis of one and two-dimensional ssNMR spectra. You will use these results to determine structural models at atomic resolution.

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Scientific background:

During DNA replication, recombination and DNA repair, double-stranded DNA frequently forms three- or four-way junctions, bubbles, flaps or broken ends with single-stranded extensions. These irregular structures must be corrected to maintain genome stability, integrity and fidelity. This task is accomplished by structure-specific endonucleases. Inactivation or malfunctioning of these enzymes causes genetic defects or cancer. The XPF family, is such a structure-specific endonuclease. In humans seven members (XPF, MUS81, ERCC1, EME1, EME2, FANCM and FAAP24) have been characterized by the presence of the ERCC4 nuclease domain. Only two of them however, XPF and MUS81, have nuclease activity mediated by the conserved core nuclease motif (ERKX3D). Their catalytic function however depends on heterodimer formation with the non-catalytic family members. For example XPF forms an obligate heterodimeric complex with ERCC1 but not with other members of the family and functions primarily in the Nucleotide Excision Repair (NER) pathway. This versatile pathway is able to detect and remove a variety of bulky DNA lesions induced by UV light and environmental carcinogens and thereby maintains genome integrity. We have previously determined the structure of various domains within this family and characterised how these proteins heterodimerize, how they bind to DNA but the catalytic reaction remains elusive and what determines the preferential heterodimer formation is also not known. Recently we have implemented an in vitro assay that functionally resembles the DNA repair reaction and assays to study protein-protein and protein-DNA interactions. These assays, in cooperation with the structural information available, permit us to study the structure-function relationship between the ability to form heterodimers, bind to DNA and perform catalysis.

Research proposal:

On the basis of available structural information and functional models design mutants in the catalytic domain of XPF, the interaction domain between ERCC1 and XPF and the DNA binding domains of ERCC1 and XPF. Clone these mutants and express the protein in a suitable expression hosts. After purification the ability to bind to DNA, to interact with the interaction partner, to perform incision reactions will be determined. For a few mutants biophysical characterisation will be performed using fluorescence methods and NMR spectroscopy. Finally using the obtained information a structural model will be made to explain the biochemical observations.

Methods:

• PCR
• Cloning
• Protein (co-)expression
• Large scale protein purification using HPLC
• Radio nuclide experiments
• Protein-protein interaction assays
• Protein-DNA interaction assays
• Site-directed mutagenisis
• DNA incision assays
• Biophysical characterisation of proteins using NMR spectroscopy
• Biophysical characterisation of proteins using fluorescence techniques

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Project:

Cyclic nucleotides (cNMPs) are important secondary messenger molecules that mediate a multitude of processes by activating several different proteins in the signaling cascade. One such family of proteins relates to ion channels. Upon binding cNMPs the channels open, which results in change in membrane potential of cells. Channels regulated by cNMPs have been extensively characterized in sensory systems like rod- and cone-photoreceptors, and excitatory cells in heart and brain, but the molecular mechanism of activation remains poorly understood. The aim of the current project is to comprehend the effects of ligand binding on channel opening.

The project involves functionally characterizing a bacterial homolog (mlCNG) of cNMP regulated channel by electrophysiological methods. The mlCNG channel serves as a useful model system, as structural reference data is available on the domains of the channel separately. Furthermore, the channel protein has also been studied with respect to ligand binding, but the activation profile by electrophysiological measurements is lacking. Electrophysiological measurements will be performed on the mlCNG channel by patch-clamp techniques on giant bacterial sphaeroplasts and liposomes. This project will be undertaken as collaborative effort between Rudolf Magnus Institute of Neuroscience, Department of Neuroscience and Pharmacology (Dr. G.M.J. Ramakers), and the NMR Spectroscopy Research Group (Prof. Dr. M. Baldus), Bijvoet Center for Biomolecular Research at the University of Utrecht.