[23] The progress in 3D structure determination of membrane proteins by X-ray crystallography and cryo electron microscopy has created an increasing demand and opportunity for in-depth mechanistic studies by magnetic resonance methods.
The view that proteins act as single entities has been replaced with the concept suggesting that dynamic reorganization of multimeric soluble complexes annotated as signalosomes is essential for signal transmission in the cell.
[34] Using mass spectrometry, a global analysis of the ubiquitinome of Salmonella-infected cells was carried out, that enabled CEF scientists to identify specific targets of bacterial ligases that are secreted into the cellular cytoplasm by the pathogens.
This isoform adopts a closed, inactive and only dimeric conformation in which both, the interaction with the DNA as well as with the transcriptional machinery is significantly reduced[47] The inhibition is achieved by blocking the tetramerization interface of the oligomerization domain with a six-stranded anti-parallel beta-sheet.
[49] These discoveries open the possibility to develop a therapy for preserving oocytes during chemotherapy which in female cancer patients usually results in infertility and the premature onset of menopause.
CEF scientists also helped to identify the molecular mechanism causing ankyloblepharon-ectodermal dysplasia-cleft lip/palate syndrome, a disease characterized by skin erosions, oral clefting abnormalities and fused eyelids, which is based on mutations in the SAM domain or in the C-terminus of p63.
[50] Complexes involved in tumorigenesis were studied by several CEF groups, including the leukemogenic AF4-MLL fusion protein[51] and RIP1-containing cytosolic complexes that are critical for the initiation and fine-tuning of different forms of cell death, i.e. apoptosis and necroptosis[52][53] Goethe University became a member of the Structural Genomics Consortium (SGC) in 2017, an international consortium and public-private partnership dedicated to the determination of structures of important proteins and the development of inhibitors and probes for biological macromolecules to be used in functional investigations.
Goethe University has also become the home and reference center for the SGC's donated probes programme, that makes small molecules no longer being further pursued by industry as drug targets freely available to researchers worldwide[54]).
CEF scientists also showed that for the guanine-sensing xpt-pbuX riboswitch of B. subtilis, the conformation of the full-length transcripts is static: it exclusively populates the functional off-state but cannot switch to the on-state, regardless of the presence or absence of ligand.
CEF scientists in collaboration with the Max Planck Institute for Biophysical Chemistry visualized the RNA Polymerase I (Pol I) in the process of actively transcribing ribosome genes in a cellular environment and solved its structure with and without nucleic acids at 3.8 Å resolution by cryo-EM.
Work by a collaboration between several CEF groups unravelled the molecular nature of Bowen-Conradi syndrome by demonstrating that the disease-causing point mutation of the ribosome biogenesis factor Nep1 impairs its nucleolar localisation and RNA binding.
In the field of optogenetics, control of membrane potential and intracellular signalling in neurons and other cells is achieved by expression of photosensor proteins, in most cases of microbial origin, e.g. ion channels or pumps, as well as light-activated enzymes.
The Gottschalk lab introduced ChR2, the light-driven Cl—pump halorhodopsin and other rhodopsins into the nervous system of the nematode C. elegans, to stimulate single neurons and correlate their function with a behavioural output.
[99][100][101] Several CEF groups joined forces not only to unravel the photocycle of ChR2 at different time scales[102] but also provided, in collaboration with the Research Centre Juelich, structural insights into ion conduction by ChR2.
[103] They also generated several mutant ChR2 versions with altered ion conductance (for example increased Ca2+-permeability in "CatCh", a Ca2+ transporting channelrhodopsin) or kinetics, representing highly useful additions to the optogenetic toolbox .
With the new rhodopsins came the observation that they represent a rather versatile family of proteins while retaining the structural scaffold of seven transmembrane helices with a retinal chromophore bound to a conserved lysine.
Synthetic retinal analogs were introduced into ChR2 or other rhodopsin tools in C. elegans, Drosophila and human cells, to change the light sensitivity, photo cycle kinetics and colour spectrum of the optogenetic actuators.
[130] New building principles for DNA-nanoarchitectures have been established in CEF[131][132][133] Also, new RNA riboswitches have been designed that can be triggered with small metabolites, exogenous molecules, or by temperature changes, as well as aptamers or self-cleaving ribozymes, which can be used to control gene expression in vivo.
A rational and minimally invasive protein engineering approach was used that left the molecular mechanisms of FASs unchanged and identified five mutations that can make baker's yeast produce short-chain fatty acids.
[143] To manipulate a protein photocycle in a directed manner, CEF groups collaborated to modify the flavoprotein dodecin at its key amino acid tryptophan with substituents carefully selected for their structural and electronic influence.
[157] CEF scientists used LSFM, for example, to image in detail the complete embryonic development of different evolutionary unrelated insects and to establish the rules and self-organizing properties of post-embryonic plant organ cell division patterns.
[165] The close collaborative teamwork of the consortium allowed tackling two major challenges in live-cell as well as single-molecule localization microscopy: efficient delivery of fluorophores across cell membranes and high-density protein tracing by ultra small labels.
The members of the Center for Biomolecular Magnetic Resonance (BMRZ) improved the sensitivity of liquid- and solid-state NMR by a spectrometer featuring dynamic nuclear polarization (DNP).
By integrating DNP-enhanced solid-state NMR spectroscopy with advanced molecular modeling and docking, the mechanism of the subtype selectivity of human kinin G-protein-coupled receptors for their peptide agonists was resolved.
[169] DNP-enhanced solid-state NMR spectroscopy enabled CEF scientists to determine the atomic-resolution backbone conformation of an antigenic peptide bound to the human ABC transporter TAP.
The results uncovered the central role of reversible conformational equilibrium in the function and regulation of an ABC exporter and established a mechanistic framework for future investigations on other medically important transporters with imprinted asymmetry.
The study also demonstrated for the first-time the feasibility to resolve equilibrium populations at multiple domains and their interdependence for global conformational changes in a large membrane protein complex.
Advantages of mass spectrometry compared to other methods like X-ray crystallography or nuclear magnetic resonance are for instance its lower limits of detection, its speed and its capability to deal with heterogeneous samples.
This method enables the observation of extremely fast chemical and biological reactions in real time involving a wide variety of molecules from small organic compounds to complex enzymes.
Their goal is to develop detailed and quantitative descriptions of key biomolecular processes, including energy conversion, molecular transport, signal transduction, and enzymatic catalysis.