Zentrum für Biomolekulare Magnetische Resonanz (BMRZ)
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Nuclear magnetic resonance (NMR) spectroscopy is a powerful and popular technique for probing the molecular structures, dynamics and chemical properties. However the conventional NMR spectroscopy is bottlenecked by its low sensitivity. Dynamic nuclear polarization (DNP) boosts NMR sensitivity by orders of magnitude and resolves this limitation. In liquid-state this revolutionizing technique has been restricted to a few specific non-biological model molecules in organic solvents. Here we show that the carbon polarization in small biological molecules, including carbohydrates and amino acids, can be enhanced sizably by in situ Overhauser DNP (ODNP) in water at room temperature and at high magnetic field. An observed connection between ODNP 13C enhancement factor and paramagnetic 13C NMR shift has led to the exploration of biologically relevant heterocyclic compound indole. The QM/MM MD simulation underscores the dynamics of intermolecular hydrogen bonds as the driving force for the scalar ODNP in a long-living radical-substrate complex. Our work reconciles results obtained by DNP spectroscopy, paramagnetic NMR and computational chemistry and provides new mechanistic insights into the high-field scalar ODNP.
The function of the p53 transcription factor family is dependent on several folded domains. In addition to a DNA-binding domain, members of this family contain an oligomerization domain. p63 and p73 also contain a C-terminal Sterile α-motif domain. Inhibition of most transcription factors is difficult as most of them lack deep pockets that can be targeted by small organic molecules. Genetic knock-out procedures are powerful in identifying the overall function of a protein, but they do not easily allow one to investigate roles of individual domains. Here we describe the characterization of Designed Ankyrin Repeat Proteins (DARPins) that were selected as tight binders against all folded domains of p63. We determine binding affinities as well as specificities within the p53 protein family and show that DARPins can be used as intracellular inhibitors for the modulation of transcriptional activity. By selectively inhibiting DNA binding of the ΔNp63α isoform that competes with p53 for the same promoter sites, we show that p53 can be reactivated. We further show that inhibiting the DNA binding activity stabilizes p63, thus providing evidence for a transcriptionally regulated negative feedback loop. Furthermore, the ability of DARPins to bind to the DNA-binding domain and the Sterile α-motif domain within the dimeric-only and DNA-binding incompetent conformation of TAp63α suggests a high structural plasticity within this special conformation. In addition, the developed DARPins can also be used to specifically detect p63 in cell culture and in primary tissue and thus constitute a very versatile research tool for studying the function of p63.
The ATP-binding cassette transporter TAPL translocates polypeptides from the cytosol into the lysosomal lumen. TAPL can be divided into two functional units: coreTAPL, active in ATP-dependent peptide translocation, and the N-terminal membrane spanning domain, TMD0, responsible for cellular localization and interaction with the lysosomal associated membrane proteins LAMP-1 and LAMP-2. Although the structure and function of ABC transporters were intensively analyzed in the past, the knowledge about accessory membrane embedded domains is limited. Therefore, we expressed the TMD0 of TAPL via a cell-free expression system and confirmed its correct folding by NMR and interaction studies. In cell as well as cell-free expressed TMD0 forms oligomers, which were assigned as dimers by PELDOR spectroscopy and static light scattering. By NMR spectroscopy of uniformly and selectively isotope labeled TMD0 we performed a complete backbone and partial side chain assignment. Accordingly, TMD0 has a four transmembrane helix topology with a short helical segment in a lysosomal loop. The topology of TMD0 was confirmed by paramagnetic relaxation enhancement with paramagnetic stearic acid as well as by nuclear Overhauser effects with c6-DHPC and cross-peaks with water.
ATP-binding cassette (ABC) transporters, a superfamily of integral membrane proteins, catalyse the translocation of substrates across the cellular membrane by ATP hydrolysis. Here we demonstrate by nucleotide turnover and binding studies based on 31P solid-state NMR spectroscopy that the ABC exporter and lipid A flippase MsbA can couple ATP hydrolysis to an adenylate kinase activity, where ADP is converted into AMP and ATP. Single-point mutations reveal that both ATPase and adenylate kinase mechanisms are associated with the same conserved motifs of the nucleotide-binding domain. Based on these results, we propose a model for the coupled ATPase-adenylate kinase mechanism, involving the canonical and an additional nucleotide-binding site. We extend these findings to other prokaryotic ABC exporters, namely LmrA and TmrAB, suggesting that the coupled activities are a general feature of ABC exporters.