NMR

The research of the NMR group is focused on the elucidation of the molecular basis of protein-DNA recognition, using NMR spectroscopic techniques, which are in many cases specifically developed for answering specific questions on the nature and extent of the interacting groups and atoms. NMR spectra provide information on the geometry of direct neighbour groups in a molecule and on the spatial proximity of hydrogen atoms in the molecule. To obtain the necessary data, several kinds of mostly multi-dimensional NMR experiments have to be performed. These data can be transformed into realistic three dimensional molecular structures by extensive computer calculations, with methods adopted from computational chemistry techniques.

The group has extensive experience in the area of structure determination of biomolecules using NMR spectroscopy. The group was involved in one of the first protein structure determinations by NMR (lac repressor headpiece, 1985) and determined the first structure of a protein-DNA complex (1987). Several contributions were made to the methodology of structural analysis by NMR (three-dimensional NMR, automated spectral analysis). The Restrained Molecular Dynamics method was worked out by the group in collaboration with W.F. van Gunsteren (then in Groningen).

Protein-DNA Interactions

For many cellular processes the recognition of DNA sequences by proteins is a fundamental event, which lies at the root of e.g. gene regulation and DNA repair. The study of the interaction processes between proteins and DNA is therefore of crucial importance for the understanding of the fine-tuning found in nature for the cascade of processes in the cell leading to the predetermined effect. Interaction processes can only be understood on a fundamental and molecular level if the three-dimensional structures of the interacting biomolecules and the resulting molecular complexes are known under conditions which are close to their natural physiological environment. In addition to obtaining three-dimensional structures in solution, NMR can be used for obtaining detailed information of the dynamics of proteins and complexes under natural physiological conditions. The group has been highly successful in this area in its study of the lac-repressor DNA binding domain or "headpiece" and its interaction with a DNA "operator" fragment. Similar studies have been done on various DNA binding domains, including those of nuclear hormone receptors and nuclear excision DNA repair complexes.

Methodology

Detailed NMR spectroscopic information can only be obtained for relatively small complexes, up to a molecular mass of 40.000 Dalton. Most protein-DNA complexes often are much larger. Therefore, usually the active domains of DNA-binding proteins are being studied. These domains are chosen in such a way that they retain the interaction mechanism of the complete molecules. A major problem in the structure determination of proteins from NMR data is the unambiguous assignment of the spectra, which are very complex due to overlap of signals. Multidimensional 3D and 4D NMR techniques, combined with advanced isotope labeling methods, exist to alleviate this problem. This enabled to obtain detailed structural and dynamical information of large protein-DNA complexes, such as of an intact lac repressor and of full nucleosomes.

NMR