Department of Molecular Biophysics (Adam Lange)

Solid-state NMR

We study protein structure and dynamics using nuclear magnetic resonance in the solid state (solid-state NMR) combined with a variety of other structural and biophysical methods. In the last decade, solid-state NMR has emerged as a powerful technique in structural biology as it gives access to structural information for systems that are insoluble or do not crystallize easily. Furthermore, the technique allows for the characterization of chemical details (e.g. protonation of side chains), the interaction with water and/or lipid molecules, and functionally important protein dynamics. For solid-state NMR investigations, samples are placed in a strong superconducting magnet (external field up to 20 T, i.e. ~400,000 times stronger than the earth’s magnetic field), spun rapidly (up to 100,000 rotations per second; magic-angle spinning), and probed by radio waves.

Membrane proteins

In our group, a major focus lies on membrane proteins. Distinct from other methods in structural biology, solid-state NMR makes it possible to study membrane proteins in native-like lipid bilayers at room temperature and under physiological buffer conditions. Current projects involve for instance non-selective cation channels such as NaK that are able to conduct both sodium (Na+) and potassium (K+) with equally high efficiency (see Figure 1). In contrast to previous crystallographic results, we could recently show that the selectivity filter of NaK in native-like lipid membranes adopts two distinct conformations that are stabilized by either Na+ or K+ ions. The atomic differences of these conformations were resolved by solid-state NMR spectroscopy and molecular dynamics (MD) simulations. We propose that structural plasticity within the selectivity filter and the selection of these conformations by different ions are key molecular determinants for highly efficient conduction of different ions in non-selective cation channels (Shi et al., Nature Communications 2018). Other membrane proteins of interest comprise the human voltage-dependent anion channel (VDAC) (Schneider et al., Angewandte Chemie 2010; Zachariae et al., Structure 2012), the histidine kinase CitA (Salvi et al., PNAS 2017), and rhomboid proteases, that initiate signal cascades by cleaving proteins within the membrane and thus releasing signal proteins that are no longer anchored.

Figure 1: The non-selective cation channel NaK was studied by solid-state NMR and MD simulations (Shi et al., Nature Communications 2018). Artwork by Barth van Rossum, FMP.

Supramolecular assemblies

Furthermore, we characterize structure and dynamics of bacterial supramolecular assemblies. For example, we have determined the structure of the bactofilin BacA by solid-state NMR. Bactofilins are a new class of cytoskeletal proteins that are involved in key cellular processes. For instance, in the human pathogen Helicobacter pylori, they are responsible for maintaining its characteristic helical cell shape, a feature required for cells to efficiently colonize the gastric mucus. We discovered that bactofilins adopt a β-helical architecture (see Figure 2), which has not been observed before for other cytoskeletal filaments. Interestingly, however, the structure bears similarities to that of the fungal prion protein HET-s. (Vasa et al., PNAS 2015 and Shi et al., Science Advances 2015). We also introduced a general hybrid approach for determining the structures of supramolecular assemblies. Cryo-electron microscopy (cryo-EM) data define the overall envelope of the assembly and rigid-body orientation of the subunits while solid-state NMR chemical shifts and distance restraints define the local secondary structure, protein fold and inter-subunit interactions. Using this approach we could determine the structure of the type-III secretion system needle of Shigella flexneri to a very high precision (Demers et al., Nature Communications 2014; see also our previous work: Loquet et al., Nature 2012).

Figure 2: High-resolution 3D structure of the cytoskeletal protein BacA (Shi et al., Science Advances 2015).

Method development

Last but not least we continue to develop new solid-state NMR methods. For instance, we recently described a protocol for the chemical shift assignment of the backbone atoms of proteins in the solid state by 1H-detected solid-state NMR. It requires a perdeuterated, uniformly 13C- and 15N-labeled protein sample with subsequent proton back-exchange to the labile sites. The sample needs to be spun at a minimum of 40 kHz in the NMR spectrometer. With a minimal set of five 3D NMR spectra, the protein backbone and some of the side-chain atoms can be completely assigned. These spectra correlate resonances within one amino acid residue and between neighboring residues (see Figure 3); taken together, these correlations allow for complete chemical shift assignment via a 'backbone walk'. This results in a backbone chemical shift table, which is the basis for further analysis of the protein structure and/or dynamics by solid-state NMR (Fricke et al., Nature Protocols 2017). More recently, we extended our approach to four dimensions (Zinke et al., Angewandte Chemie 2017) and additionally exploited sophisticated methyl labeling schemes for the detection of intra- and intermolecular distance restraints (Zinke et al., ChemPhysChem 2018). Those proton-detected solid-state NMR strategies will be employed to study membrane-integrated proteins.

Figure 3: Assignment of complex protein spectra by proton-detected 3D solid-state NMR (Fricke et al., Nature Protocols 2017).

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Leibniz-Forschungsinstitut für Molekulare Pharmakologie im Forschungsverbund Berlin e.V. (FMP)
Campus Berlin-Buch
Robert-Roessle-Str. 10
13125 Berlin, Germany
+4930 94793 - 100 
+4930 94793 - 109 (Fax)
info(at)fmp-berlin.de

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