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FACULTY OF BIOLOGY, CHEMISTRY & EARTH SCIENCES

Biochemistry IV - Biophysical Chemistry - Prof. Dr. Janosch Hennig

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Research

With well-equipped molecular and biochemistry labs and access to a full range of high-flield solid- and liquid-state NMR spectrometers (600 MHz, 700 MHz, 900 MHz and 1 GHz) located at the NBNC (Northern Bavarian NMR Center), the Chair of Biochemistry IV is engaged in many areas of research in molecular and structural biology relevant to human disease and to the understanding of basic biological processes. Frequent access to synchrotron beam lines and cryo-EM microscopes enables our approach of integrative structure modelling of biomolecular complexes.
Read more about NMR structure determinationHide

NMR structure determination

Solution state NMR spectroscopy allow the observation of numerous spin interactions which carry structural information of biomacromolecules. The chemical shift of protein signals strongly depends on the secondary structure, scalar couplings across three bonds gives information about the central dihedral angle. Cross relaxation between protons causes cross signals in NOESY spectra which strongly depends on the internuclear distances. These distance data is by far the most important information for structure determination. In addition residual dipolar couplings and hydrogen bonds contribute to the amount of structural information. Using solution state NMR spectroscopy we have determined numerous protein structures as well as protein-protein complexes.

Read more about NMR as tool for analyzing protein dynamicsHide

NMR as tool for analyzing protein dynamics

Proteins are not rigid molecules and often dynamic structural changes are required for their biological function. NMR spectroscopy is a powerful method for analyzing protein dynamics on a wide range of timescales. Different mobilities on timescales faster than the rotational correlation time (ps-ns) can be investigated by 15N relaxation rates. Chemical exchange processes on the μs-ms timescale can be addressed by relaxation dispersion measurements. Chemical Exchange Saturation Transfer (CEST) is a useful method to detect dynamics on the ms-s timescale even if the excited states are low populated. Measuring hydrogen exchange allows the characterization of local and global stabilities of a folded protein.

Read more about NMR Data processingHide

NMR Data processing

Modern NMR spectrometer show a very high sensitivity. As a consequence the duration for multidimensional NMR experiments is not determined by the signal accumulation for sufficient signal/noise (sensitivity limit) but the overall experiment time is determined by recording the number of data points along the indirect dimensions (sampling limit). Leaving out a majority of data points (non uniform sampling, NUS) and reconstructing the spectrum by more sophisticated methods than the traditional Fourier Transform offers significant savings in NMR time. We apply a self written software based on the Iterative Soft Threshold reconstruction method. Using this approach triple resonance experiments can be run in a few minutes, depending of the sensitivity of a given sample.

Research AG Hennig

The Hennig group employs integrated structural biology (nuclear magnetic resonance (NMR) spectroscopy, X-ray, small-angle scattering and cryo-electron microscopy) to investigate the molecular mechanisms underlying translation regulation and ribonucleoprotein complex assembly.

Previous and current research

Dosage compensation is an essential molecular process in sexually reproducing organisms, which compensates the imbalance of number of sex chromosomes between the sexes. Although these processes can be quite different between species, recent research shows that all have common mechanisms. One of which is the involvement of complexes between proteins and different RNA molecules, mRNA and long non-coding RNAs, often involved in phase separation.

In Drosophila, we study both, the female and male side. In males, we want to understand how exactly the long non-coding RNAs RoX1 and RoX2 are remodelled to allow assembly of the dosage compensation complex (or MSL complex) on the single male X chromosome to achieve 2-fold hypertranscription. This hypertranscription would be lethal in females. Instead, the female-specific protein sex-lethal (Sxl) binds to the mRNA of the MSL complex’ rate-limiting component MSL2, to prevent its translation. The highly conserved protein Upstream-of-N-Ras (Unr) is recruited by Sxl to the same site on the mRNA (Figure1) and, together with Hrp48, essential for translation repression of msl2 mRNA (Figure 2). The male MSL complex is also conserved in humans, where it is regulating autosomal compensation. All RNA binding proteins regulating translation in female flies are conserved in humans, and Unr for example is highly expressed in certain cancer cell lines.

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Another main project of the lab revolves around novel RNA binding proteins, meaning proteins, which do not feature a classical RNA binding domain (like RRM, CSD, KH or dsRBD domains), but have been shown to bind single-stranded RNA in mRNA interactome capture. Of special interest to us are RNA binding E3 ligases of the tripartite motif (TRIM) protein family. Here we want to understand how RNA binding is connected to these proteins’ main biochemical function: ubiquitination. Based on our data, we hypothesize that some TRIMs bind to mRNA and regulate translation by ubiquitinating components of translation complexes. Other TRIM proteins and their ubiquitination function seems to be actually regulated by RNA, which we could recently show for TRIM25 (Haubrich et al., BioRxiv, 2020, Figure 3).

Future projects and goals

Our ultimate goals are to obtain high resolution structures of these large protein-RNA complexes validated by biochemical and cell biological experiments to get a detailed molecular understanding of these essential mechanism. To this end, we employ all available structural biology methods (NMR, X-ray crystallography, cryo-EM and small-angle scattering) and more.

We also collaborate on many exciting projects within and outside of the University of Bayreuth, where we help out with our NMR and integrative structural biology expertise.

Research AG Wöhrl

Structure and function of G-protein coupled receptors

G-protein coupled receptors (GPCRs) are integral membrane proteins and represent one of the largest families of proteins in mammals. More than 800 genes in the human genome code for GPCRs. GPCRs consist of 7 transmembrane helices and participate in diverse physiological functions. The extracellular ligands include ions, photons, amines, peptides, nucleotides, odorants etc. Signal transduction is achieved via binding of various heterotrimeric G-proteins, consisting of Gα, Gβ, and Gγ subunits, or β-arrestins at the intracellular loops of the receptors, thus activating a cascade of intracellular signalling proteins which in turn result in a specific physiological response.
​GPCRs are widely used as drug targets and various approaches to develop new ligands that are active as drugs are being explored. Thus, our understanding of the mechanism of integration and folding of the receptors into the membrane as well as signal transduction and drug action need to be improved.

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Although the general cascade of events e.g. for the cAMP signaling pathway is well known, the processes on a molecular level, i.e. the conformational changes that occur upon ligand binding and transfer of the information into the interior of the cell still need to be elucidated.

Signal transduction by GPCRs via the cAMP pathway and an activating G-protein. Upon binding of an extracellular ligand, a conformational change in the GPCR leads to exchange of GDP for GTP and dissociation of the α subunit of the G-protein from Gβγ and the GPCR. GTP-bound Gα can then associate with an effector protein and thus activate e.g. the membrane bound adenylyl cyclase which catalyzes the formation of the second messenger molecule cAMP from ATP. cAMP activates downstream proteins in the signal cascade which in turn finally leads to a cellular response.  

We are working on the human inhibitory dopamine receptors D2 and D3 to learn more about their biophysical and biochemical properties and to shed light on conformational changes triggered by ligand binding in order to transduce a signal into the interior of the cell. D2 and D3 are known to bind and mediate the functions of dopamine, an important neurotransmitter in the central nervous system.
The goal of our project is the investigation of ligand-receptor interactions and dynamics of the GPCRs using different biochemical and biophysical methods. We develop tools for mutagenesis resulting in engi-neered receptor variants, as well as their stable expression and purification. Biophysical experiments with membrane proteins are still very challenging since the receptor has to be reconstituted in a lipid bilayer, e.g. vesicles or nanodiscs for correct folding and functionality. The future goal is the analysis of ligand‐receptor interactions and dynamics of GPCRs by biophysical methods, e.g. nuclear magnetic resonance (NMR), electron paramagnetic resonance (EPR) or fluorescence.

Structure, stability and physiological function of PR-10 allergens

Globally about 250 million people suffer from food allergies. Many risk factors have been suggested to be associated with the development of allergies, i.e. environmental pollution, tobacco smoke, climate change, altered human gut flora due to nutritional changes etc. Although allergies in developed countries are still on the rise, no true treatment is available. The immune system of people suffering from food allergies combats small proteins existent in pollen, fruit or vegetables that are harmless. Antigen-specific IgE antibodies, together with one of the major effector cells of allergy, the mast cells are crucial for the development of the acute manifestations of these allergic disorders.

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The so-called pathogen-related (PR-10) proteins are causative agents of food allergies. They are found in pollen of birch, alder and hazel, as well as in vegetables or fruits like carrot, celery, pear etc. In birch the most important allergy causing protein is the PR-10 protein Bet v 1. Among birch pollen allergic patients up to 70% develop so-called cross allergies to Bet v 1-homologue food allergens found in fruits or vegetables. These cross-allergies are caused by IgE antibodies that bind to similar or identical structural epitopes on the different allergens which share high structural homology.

Overlay of PR-10 allergen structures. Cor a 1.0401 (black), Bet v 1.0101 (blue, 1BV1), Fra a 1E (green, 2LPX) , Gly m 4 (yellow, 2K7H) and Pru av 1 (pink, 1E09)

Although they are present in many plants, knowledge on their functions is scarce. Our goal is to understand the structure and biological function of PR-10 proteins. To elucidate the function of PR-10 proteins we identify their natural ligands. For these purposes we isolate allergens from natural sources or use recombinantly expressed proteins.  We apply various protein purification techniques, ligand extraction and use NMR, HPLC, mass spectrometry, CD spectrometry and biochemical and molecular biology techniques to investigate protein structure, ligand binding and to identify the corresponding ligands.

Identification of genes involved in ulcerative colitis carcinoma

Ulcerative colitis (UC), together with Crohn ‘s disease (CD), is one of the most common chronic inflammatory bowl disease (IBD). Symptoms include chronic diarrhea and abdominal pain, as well as the appearance in phases.

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Patients with UC have increased risk of developing colorectal cancer (CRC). On average, about five percent of UC patients develop CRC, therby the risk increases with the duration of the disease. If CRC is diagnosed in a patient with UC, it needs to be determined whether it is a sporadic or a colitis-associated carcinoma since further treatment depends on its origin. While sporadic CRCs can be ablated locally, colitis-associated CRCs require removal of the colon along with the rectum (proctocolectomy). At present, histological material from biopsies is investigated to distinguish the two CRC types. However, some cases are difficult to judge microscopically. Therefore, in a collaborative effort of the Department of Pathology of the Bayreuth Clinic and the Chair of Biopolymers at the University of Bayreuth this project aims at the identification of (a) genetic marker(s) that unequivocally allow to discriminate between the two forms of CRC.

Since DNA methylation is important for gene expression, we analyze and compare the DNA methylation patterns of healthy persons, patients with UC, but without CRC, and patients with sporadic or colitis-associated CRC. In addition, patients are screened for mutations in tumor-relevant genes. 

Research AG Knauer

Transcription regulation in prokaryotes and eukaryotes

Transcription is the conversion of genetic information stored in DNA into RNA and thus a central process in all cells. Cellular genomes are transcribed by multisubunit, DNA-dependent RNA polymerases (RNAPs), which are evolutionary related in the three kingdoms of life. Every step in the transcription cycle, i.e. initiation, elongation, and termination, is highly regulated by a multitude of transcription factors that bind to RNAP to modulate its function, sometimes by forming huge multicomponent nucleic acid:protein complexes.
Using microbiology, biochemistry, biophysics, and structural biology methods we aim to understand the fundamentals of transcription regulation in pro- and eukaryotes in molecular detail.  Our findings do not only contribute to expand the knowledge on transcription, but reveal new concepts in the structure-function relationship and protein folding, and they may provide the basis for the rational development of new drugs that are urgently needed to counteract the ever increasing problem of antimicrobial resistance.

Currently, we are working on the following projects:

The role of NusG proteins and their paralogs in transcriptionHide

The role of NusG proteins and their paralogs in transcription

NusG proteins stand out among all transcription factors as they constitute the only class of transcriptional regulators that is universally conserved (called Spt5 in archaea and eukaryotes, DRB-sensitivity-inducing factor (DSIF) in humans). NusG proteins have a modular structure and consist of at least two domains that are connected via a flexible linker: an N-terminal domain (NTD) with conserved, mixed alpha/beta topology and a C-terminal domain (CTD) that adpots a five-stranded, antiparallel beta-barrel harboring a KOW motif. Like bacterial NusG proteins archaeal Spt5 proteins also consist of an NTD and a CTD, but additionally form heterodimers with Spt4. In eukaryotes the inreased regulatory complexity is reflected by a more elaborate architecture as an unstructured N-terminus is followd by an NTD, several KOW domains and a mobile C-terminal repeat region.

The NusG/Spt5-NTD binds to RNAP increasing its processivity while the CTD is used to couple RNAP to various interaction partners and concurrent processes. The regulation of trancription by NusG proteins relies on the concerted assembly of multi-component nucleic acid: protein complexes and autoinhibition. In addition to housekeeping NusGs that regulate the majority of the genes, many species also encode specialized NusG paralogs, which modulate the expresison of a subset of genes and sometimes even act orthogonally to NusG. The best studies example is E. coli RfaH, whose action is restricted to genes that have an operon polarity supressor (ops) site in the untranslated leader region. RfaH regulation involves reversible fold switching of its CTD that goes beyond all schemes known from metamorphic proteins, why RfaH is the founding member of the family of transformer proteins and may serve as model system to gain deeper insights into the understanding of structure-function relationship and protein folding. So far, the molecular mechanism of recruitment of RfaH and the molecular basis why RfaH, in contrast to ubiquitous NusG, is a transformer protein, are not understood. Additionally, it is unclear if other specialized NusG paralogs, which are often involved in pathogenesis, use similar regulatory mechanisms.

The structural basis for λQ-dependent antiterminationHide

The structural basis for λQ-dependent antitermination

Transcription is a discontinuous process and RNAP pauses from time to time. These pauses allow efficient regulation by a plethora of mechanisms that  may lead to continued transcription or prolonged pausing, the latter being the precursor of termination. Generally, termination occurs at termination sequences via two mechanisms: In intrinsic termination the nascent RNA forms a short GC-rich hairpin structure that is followed bei a polyU sequence. In Rho-dependent termination the termination factor Rho, a homohexameric ATPase, binds to a Rho utilization site, translocates towards the RNAP, and finally induces termination. Under certain circumstances termination is suppressed in a process called antitermination (AT). AT mechanisms were first discovered in context with bacteriophage λ, where they control the expression of early and late genes. &lamda; uses two different AT mechanisms, the λN- and the λQ-dependent AT, the first of which is the best studied. λQ is recruited to RNAP during initiation or early elongation via a Q binding element (QBE) in the upstream DNA. The molecular basis of recruitment, recognition by RNAP, which other factors are involved, and how it suppresses termination signals many bases downstream of the recruitment site is elusive.

The regulation of ribosomal RNA synthesisHide

The regulation of ribosomal RNA synthesis

In a bacterial cell ribosomal RNA (rRNA) and transfer RNA /tRNA) make of more than 95% of the total RNA and a major frcation of the energy of a cell is invested into ribosome synthesis. Thus, synthesis, cleavage, and maturation of rRNA as well as their assembly with ribosomal proteins is tightly controlled. The proper and efficient synthesis of ribosomal RNA relies on the supression of certain termiantion signals (antitermination) and the correct folding of the RNA. These processes involve N-utilization substances A, B, E, and G as well as ribosomal protein S4 and the inositol monophosphatase SuhB. However, the structural basis of these processes remains elusive.

The rational development of drugs that inhibit bacterial transcriptionHide

The rational development of drugs that inhibit bacterial transcription 

Antimicrobioal resistance is one of the major health problems worldwide as an ever-increasing number of bacterial strains become resistant to standard antibiotics and new drugs are lacking. To counteract this scenario we plan to contribute to the targeted development of inhibitors of bacterial DNA:RNA transcription. We use the atomic information we gain in the regulation of bacterial transcription to identify new targets and subsequently develop drugs that specifically disturb the fine-tuned regulatory transcription network.


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