The Research Training Group "Protein Complex Assembly" (GRK 2223) is funded by the German Research Foundation (DFG). We started our first founding period in October 2016.
The molybdenum cofactor is a unique pterin derivative catalyzing two electron transfer reactions in the active site of molybdenum enzymes (Mo-enzymes). Structure based biology revealed the molecular mechanism of the plant molybdenum insertase Cnx1E and identified three of its four subdomains being involved into catalyzes. Cnx1E mechanistic work suggests that Cnx1E subdomain IV mediates interactions within the Moco biosynthesis complex. The aim of this project is to decipher the molecular interactions occurring within the Moco biosynthesis complex during and upon Moco synthesis. A broad and interdisciplinary approach will be used to decipher this network.
Prof. Dr. Ralf-R. Mendel
+49531-391-5870
r.mendel(at)tu-braunschweig.de
Dr. Tobias Kruse
+49531-391-5873
t.kruse(at)tu-braunschweig.de
Kevin Oliphant
+49531-391-5866
k.oliphant(at)tu-braunschweig.de
Dr. Ahmed Adel Ibrahim Hassona Hassan
Dr. Thomas Hercher
Franziska Lehne
The molybdenum cofactor (Moco) is synthesized by a conserved four-step pathway that has been deciphered in detail on molecular and biochemical level by the Mendel group in eukaryotes. Presently, we focus on subcellular localization of the involved proteins as well as on quantitative details of their interactome (under in vivo-conditions) upon forming the Moco biosynthesis complex. We hypothesize the Mo insertase Cnx1 to be positioned near the place where molybdate enters the cell to guarantee an efficient and fast transfer and incorporation of the ion into the molybdopterin scaffold of the cofactor. Therefore in this project, we aim at studying the spatial and the temporal role of the actin cytoskeleton for the formation, dynamics and stabilization of the Moco biosynthesis complex. We assume that the anchoring of the complex is of vital importance not only for the formation of the complex but also for its positioning near the three types of molybdate transporters.
Prof. Dr. Robert Hänsch
+49531-391-5867
r.haensch(at)tu-braunschweig.de
Jan-Niklas Weber
+49531-391-5875
jan-niklas.weber(at)tu-braunschweig.de
Dr. Christin-Kirsty Baillie
Dr. Rieke Meinen
We apply various tools based on fluorescence and spectroscopy to understand the functioning of higher ordered complexes and the well-balanced temporal as well as spatial coordination of subunit-assembly and disassembly. Prominent examples are two-photon fluorescence correlation spectroscopy, fluorescence polarization experiments or Förster energy transfer type techniques. All these fluorescence techniques can provide detailed information about the assembly and disassembly of heterogonous or highly ordered protein complexes and have distinct advantages and applications ranges. We will apply these techniques for the investigation of important protein assemblies such as the Moco biosynthesis complex, actin networks or the denitrification respirasome.
Prof. Dr. Peter Jomo Walla
+49531-391-5328
p.walla(at)tu-braunschweig.de
Rainer Matis
+49531-391-55244
r.matis(at)tu-braunschweig.de
Julia Nowak
+49531-391-55228
j.nowak(at)tu-braunschweig.de
Dr. Frida Kage
Dr. Andreas Albrecht
This project addresses protein assembly during lamellipodia formation. These structures are made by continuous actin filament branching off so-called mother filaments, mediated by Arp2/3 complex, a seven subunit protein assembly including two actin-related proteins serving as nucleus of the newly generated daughter filament. In addition, Arp2/3 activity in cells is tightly controlled by conformational changes, triggered by interaction with nucleation promoting factors (NPFs) that come in multiple flavors and with subcellular specificity in mammals. Lamellipodial Arp2/3 complex activation is likely restricted to the WAVE regulatory complex (WRC), operating downstream of the small Rho-GTPase Rac, although details of WRC regulation and assembly as well as the precise functions of its five subunits remain largely unknown. This project will uncover WRC regulation and recruitment mechanisms to the leading edge membrane during lamellipodia protrusion. It will also dissect the relative contributions to Rac-dependent protrusion of other small GTPases, including RhoG, Cdc42 and Arf.
Prof. Dr. Klemens Rottner
+49531-391-3256
k.rottner(at)tu-braunschweig.de
Hermann Döring
+49531-391-3258
hermann.doering(at)tu-braunschweig.de
Dr. Frida Kage
Dr. Matthias Schaks
Vanessa Dimchev
During anaerobic respiration nitrate (Nar), nitrite (Nir), nitric-oxide (Nor) and nitrous-oxide (Nos) reductases, in cooperation with accessory proteins, catalyze the reaction cascade of NO3- → NO2- → NO → N2O → N2. We utilized membrane interactomics in combination with electron microscopy co-localization studies to identify the integral membrane proteins NorC, NorB and NosR as major assembly platform for the denitrification supra-complex. Our project aims at the determination of the exact molecular basis of the observed multiple protein-protein interactions at the amino acid sequence level. The interacting domains of two crosslinked proteins can be identified via a combination of genetics and quantitative proteomics as demonstrated for the interaction of NirS with FliC before. Protein-protein interactions of interest are: NosR/NorB, NosR/NorC, NarH/NorC, NirF/NorC, NirQ/NorC, NosZ/NorR, NosL/NorC.
Prof. Dr. Dieter Jahn
+49531-391-55101
d.jahn(at)tu-braunschweig.de
Dr. Martina Jahn
+49531-391-5815
m.jahn(at)tu-braunschweig.de
Juan José Vargas Guerrero
+49531-391-5831
j.vargas-guerrero@tu-braunschweig.de
Dr. Toni Mingers
Dr. Katrin Müller
Dr. Kim Eileen Rennhack
The assembly of respiratory complexes in the respirasome is considered as an optimization of the electron transport chain. Now, it has been recognized that electron leakage plays an important functional role in T cells, which undergo profound metabolic changes along activation. Electrons "leak" from respiratory complexes and trigger the generation reactive oxidative species (ROS) in the mitochondria (mt). Interestingly, mtROS directly supports T cell activation constituting a novel model for our understanding of respirasome dynamic. We hypothesize that mtROS production in activated T cells affects the organization and dynamics of the respirasome and we will analyze redox modifications and co-occurring protein turnover by proteomics. Ultimately, this project should complement our knowledge of respirasome "plasticity" and the risk of mitochondrial dysfunctions in T cells.
Prof. Dr. Lothar Jänsch
+49531-6181-3030
lothar.jaensch(at)helmholtz-hzi.de
Nicole Amsberg
+49531-6181-3047
nicole.amsberg(at)helmholtz-hzi.de
Tina Rietschel
+49531-6181-3028
tina.rietschel(at)helmholtz-hzi.de
Dr. Daniel Meston
Identification und functional characterization of very small proteins with less than 100 amino acids is an emerging field in microbiological research and it is assumed that tens if not hundreds of these proteins are synthesized in a bacterial cell. In previous studies this group of proteins has been clearly overlooked due to challenges in their prediction and detection. This project aims at identifying small membrane associated proteins that might be integrated into the respirasome in Pseudomonas aeruginosa and their direct interaction partners and at elucidating the role of these small proteins within this complex. For identification of small membrane proteins classical membrane preparation procedure in combination with GeLC-MS/MS analysis will be used. In addition, new P. aeruginosa protein databases containing small proteins with up to 100 aa in length will be generated by using different algorithms. By this way we want to establish a very comprehensive proteome map for small proteins integrated into or even associated with the membrane in P. aeruginosa. Afterwards the membrane associated proteome will be analyzed and quantified under aerobic as well as anaerobic conditions. Small proteins that are differently synthesized under these conditions will be further characterized in terms of localization, function and interacting proteins.
Prof. Dr. Susanne Engelmann
+49531-6181-3041
susanne.engelmann(at)helmholtz-hzi.de
Alexander Beckmann
+49531-6181-4024
alexander.beckmann(at)helmholtz-hzi.de
Jessica Grube
+49531-6181-4023
Jessica.Grube(at)helmholtz-hzi.de
Dr. Erik Lehmann
Dr. Julia Bosselmann
Nicole Beier
The insertion of complex cofactors into enzymes often requires helper proteins that not only participate in the insertion process itself but may also be involved in biosynthesis and transport of these molecules. Nitrite reductase NirS, a central enzyme in nitrate respiration of microorganisms including pathogens such as Pseudomonas aeruginosa, contains two types of heme cofactors, namely a heme c and a heme d1. While heme c depends on the well-studied cytochrome c maturation system, much less is known about the processing of heme d1. Previous work in our groups has shown that heme d1 is escorted and processed by NirF and NirN, who not only organize its insertion into NirS but are also involved in a terminal oxidation step. Work in this project entails structure determination of these proteins and of their complexes as well as their biophysical characterization.
Prof. Dr. Wulf Blankenfeldt
+49531-6181-7000
wulf.blankenfeldt(at)helmholtz-hzi.de
Christian Behlendorf
+49531-6181-7023
christian.behlendorf(at)helmholtz-hzi.de
Steffi Henke
+49531-6181-7004
steffi.henke(at)helmholtz-hzi.de
Dr. Thomas Klünemann
Dr. Maurice Günther Diwo
Cytochrome P450 monooxygenases are ubiquitously occurring enzymes that play important roles in steroid biosynthesis, the detoxification of xenobiotics as well as the utilization of carbon sources. In multi-component cytochrome P450 monooxygenases, protein-protein interaction is crucial for efficient electron transfer from the cofactor NAD(P)H to the heme-containing monooxygenase component - a prerequisite for catalytic activity. Within the project, we are studying a novel actinobacterial redox partner system, which uses a [3Fe-4S]-cluster ferredoxin instead of the typical [2Fe-2S]-cluster ferredoxins found in many other three-component P450s. We are investigating the ability of this ferredoxin to interact with different types of cytochrome P450 monooxygenases to support catalysis. We are specifically interested in (1) identifying surface residues that are important for complex formation by means of protein engineering and ITC, and (2) elucidating possible electron transfer pathways within the proteins using spectroscopic and bioelectrochemical methods. As the overall aim, we will address the question if [3Fe-4S]-cluster ferredoxins, which are part of many actinobacterial three-component P450 systems, present highly specific or more universal electron transfer partners for bacterial P450 monooxygenases.
Prof. Dr. Anett Schallmey
+49531-391-55400
a.schallmey(at)tu-braunschweig.de
Nils Daniel
+49531-391-55405
n.daniel(at)tu-braunschweig.de
Margarita Stirz
+49531-391-55400
m.stirz(at)tu-braunschweig.de
Dr. Jhon Alexander Rodriguez Buitrago
Dr. Hauke Voß
Depending on the cellular molybdenum cofactor (Moco) demand, the Moco biosynthesis machinery requires being up- or down-regulated. In plants and other autotrophic organisms nitrate reductase (NR) is the major Moco user. Under N-derepressing (i.e. NR demanding) conditions, nitrate reductase expression (fungi) and/or activity (plants) is enhanced in these organisms. Contrary the expression of Moco biosynthesis genes is strongly down regulated (fungi), going along with the post translational up-regulation of Moco biosynthesis activity. The aim of this project is to decipher the nature of post-translational modification(s) which cause the up regulation of Moco synthesis activity. To reveal post translational modifications (PTM) of Moco biosynthesis enzymes we will carry out a proteomic approach. Proteomic data will be combined with available structural and mechanistic knowledge to shed light on the molecular function(s) of Moco biosynthesis PTM(s).
Prof. Dr. Simon Ebbinghaus
+49531-391-55243
s.ebbinghaus(at)tu-braunschweig.de
Janine Fichtner
The host colonization process by P. aeruginosa relies on the following sequential steps: i) planktonic cells migration towards a desired niche by flagellar motility, ii) adhesion onto the host by means of flagella or pili, iii) loss of flagella as a result of signal transduction and secretion of exopolysaccharide matrix, iv) development of a mature biofilm onto the CF mucosa lung and v) final detachment and swimming to another environment provoked by environmental cues (PMID: 12458153). The decreasing O2 gradient generated as a result of the thick mucosa of a cystic fibrosis transmembrane conductance regulator (CFTR) mutated CF lung and the biofilm density poses an adverse scenario to bacteria that P. aeruginosa circumvents by its versatile metabolism, which encompasses nitrate/nitrite respiration, arginine and pyruvate fermentation as predominant energy generating routes. On the other hand, a successful colonization of the human host and penetration through the thick mucus extensively depends on the construction of a highly developed flagellar machinery, which endows this monoflagellated bacterium with both motility and adhesion capacities. In a previous work our group revealed that FliC, although quantitatively scarce, was distributed alongside the extracytosolic region and not exclusively located at the pole where the sole flagellum of P. aeruginosa is constructed (Borrero et al 2015). The periplasmic location of FliC was discovered to take place in a triad-manner with two other proteins, the periplasmic nitrite reductase involved in denitrification and the presumably cytosolic ATP-dependent molecular chaperone DnaK. Beyond identifying the interlaced function of the nitrite reductase in swimming motility and flagellar assembly (Borrero et al 2015) no further characterization of a potentially alternative role of the DnaK besides its well-understood chaperone-like function in the flagellar export, construction or functionality has been pursued. In a recent study we monitored the location of DnaK and FliC throughout the different fractions and organelles of P. aeruginosa by harnessing scanning and transmission electron microscopy (S/TEM) coupled with single and double immunogold labelling, confocal microscopy and immunofluorescence and mass spectrometry (LC-MS/MS). Moreover, we deepened into the interaction model of DnaK-FliC by employing SPOT-membrane arrays pinpointing the interacting domains of both proteins leading to ultimately infer a docking model. We irrefutably demonstrate by different techniques an unexpected DnaK location at the flagellar filament co-localizing with FliC monomers and it to be embedded in a vesicle-like membranous structure. The aim of the follow-up project is to explore the dynamics of the protein-protein interactions underlying the flagellar and vesicle formation of P. aeruginosa employing diverse interactomics methods. Holistic characterization of the bacterial living system will be conducted using a multi-omics approach.
Hassett, D. J., Cuppoletti, J., Trapnell, B., Lymar, S. V., Rowe, J. J., Yoon, S. S., Hilliard, G. M., Parvatiyar, K., Kamani, M. C., Wozniak, D. J., Hwang, S-H., McDermott, R. R. & Ochsner, U. A. (2002) Adv. Drug Deliv. Rev. 54(11), 1425-1443.
Prof. Dr. Dieter Jahn
+49531-391-55101
d.jahn(at)tu-braunschweig.de
Viktoria Otto
+49531-391-5804
vi.otto(at)tu-braunschweig.de
Ilka Pusch
+49531-391-5890
i.pusch(at)tu-braunschweig.de
Dr. José Manuel Borrero-de-Acuña
Our knowledge of the interaction of motility and energy generation and many other multiprotein complexes in the periplasm of gram negative bacteria is very limited. A denitrification megacomplex has been suggested to be involved in Pseudomonas aeruginosa anaerobic respiration and recent studies also indicate unexpected crosstalk between the denitrification complex and the flagellar transport system through interaction of NirS-DnaK and the flagellar protein FLiC. We will develop 2D and 3D superresolution fluorescence microscopy for the study of periplasmic proteins and membrane complexes in Pseudomonas aeruginosa. The largely periplasmic and membrane spanning localization impedes analyses with classical molecular biological methods but facilitates staining with organic fluorescent dyes via SNAP-tags, Halotags or immune fluorescence from outside. With the labeled complexes, we will localize, track and count denitrification complex components under aerobic and unaerobic conditions and we will monitor the coupling between the denitrification and the motility system.
Former PhD-Student:
Rainer Matis
In this project the electron transfer chain of soluble cytochrome P450 enzymes is investigated. P450s catalyse selective oxygenation reactions of many organic compounds. Such transformations lead to hardly available compounds of technical relevance, and optimization of these processes is therefore of great commercial interest. For many soluble bacterial and mitochondrial P450s the upstream electron donor protein is not known, and ferredoxins from other P450 enzymes are employed instead in technical applications. This leads to occasional problems with electron transfer efficiency and thus with catalytic performance.
The project aims at the investigation of the cofactor-driven protein assembly and control of redox potential to optimize electron transfer processes between the soluble bacterial monooxygenases CYP154C5 and CYP154H1 and the robust ferredoxin from T. fusca which is available from the Schallmey group (P10). The necessary variation of the cofactor will be employed by making use of cofactor exchanged cytochrome P450 variants, which are generally accessible by a co-induction approach.
Former PhD-Student:
Anjali Anilkumar
Depending on the cellular molybdenum cofactor (Moco) demand, the Moco biosynthesis machinery requires being up- or down-regulated. In plants and other autotrophic organisms nitrate reductase (NR) is the major Moco user. Under N-derepressing (i.e. NR demanding) conditions, nitrate reductase expression (fungi) and/or activity (plants) is enhanced in these organisms. Contrary the expression of Moco biosynthesis genes is strongly down regulated (fungi), going along with the post translational up-regulation of Moco biosynthesis activity. The aim of this project is to decipher the nature of post-translational modification(s) which cause the up regulation of Moco synthesis activity. To reveal post translational modifications (PTM) of Moco biosynthesis enzymes we will carry out a proteomic approach. Proteomic data will be combined with available structural and mechanistic knowledge to shed light on the molecular function(s) of Moco biosynthesis PTM(s).
Former PhD-Student:
Dr. Simon Wajmann