Methods employed in our lab
In our lab we use molecular biology as well as biochemical and biophysical techniques to characterize proteins and their interaction partners in molecular detail. We apply a variety of methods, including targeted mutagenesis, protein production and purification (Escherichia coli and insect cells), in vitro transcription and translation, nanodiscs, qPCR, analytical gel filtration, circular dichroism (CD) spectroscopy, fluorescence spectroscopy, and HPLC/FPLC. To explore the molecular structure of proteins, their complexes and interactions we utilize solution-state nuclear magnetic resonance (NMR) spectroscopy and X-ray crystallography.
Some insights into NMR and X-ray crystallography
Protein NMR is a spectroscopic technique used to obtain the three-dimensional structure of a particular protein in solution at atomic resolution. It is also a powerful technique to explore the dynamics of proteins and their interactions with various binding partners (small molecules, nucleic acids, other proteins). Different spectra reveal detailed information about the chemical environment, dynamics and reaction state of a molecule.
The environment of atomic nuclei within a molecule shields an external applied magnetic field at the position of the nuclei. This modulates the resonance frequency of the corresponding spin in an NMR spectrometer (chemical shift). Therefore many similar spins within a protein can be distinguished making NMR spectroscopy a powerful technique for structure determination. A variety of interactions of the spins with the magnetic field and with other spins allows determination of three-dimensional structures, characterization of the dynamics on various timescales, and investigation of molecular interactions of proteins and other molecules.
Additionally, we use X-ray crystallography for the determination of the three-dimensional structure of proteins and their complexes. A highly pure sample is crystallized and the resulting crystal is exposed to an X-ray beam. The crystalline structure causes the X-ray beam to diffract into many specific directions. Analysis of the diffraction pattern allows the calculation of an electron density map into which a structural model of the target biomacromolecule can be built.
Structures determined recently in our lab
Solution structure of the hazelnut allergen Cor a 1.0401 (pdb: 6GQ9). Cartoon representation of the average of the 10 lowest energy solution structures of Cor a 1.0401. The structure reveals the protein fold characteristic for members of the family of pathogensis related (PR-10) proteins. It consists of a seven-stranded antiparallel β-sheet, two short α-helices arranged in V-shape and a long C-terminal α-helix encompassing a hydrophobic pocket. The strong structural similarity with the major birch pollen allergen Bet v 1 explains why many people allergic to birch pollen suffer from allergic cross reactions after consumption of raw hazelnuts.
Crystal structure of the transcription factor RfaH (pdb: 5OND) from Escherichia coli in complex with the operon polarity suppressor (ops) element, a short piece of single stranded DNA. This structure reveals the molecular basis of sequence-specific recruitment of RfaH to the transcription elongation complex pausing at an ops site. For more information: https://doi.org/10.7554/eLife.36349
NMR structures of the KOW4-x domain and the tandem domain KOW6-7 (pdb: 6EQY, 6ER0) of the human transcription elongation factor DSIF. DSIF is highly conserved throughout all kingdoms of life and plays multiple roles during transcription. It comprises several KOW domains which fullfil different functions in modulating RNA polymerase II within the transcription complex. Whereas KOW4 interacts preferentially with single stranded RNA and binds weakly to RNA polymerase II subunits Rpb4/7, the role of KOW6-7 remains elusive. For more information: https://doi.org/10.1038/s41598-018-30042-3