JavaScript Menu, DHTML Menu Powered By Milonic

Research at the department of NMR spectroscopy

The research of the Department of NMR Spectroscopy is focused on the elucidation of the molecular basis of protein-DNA recognition, using NMR spectroscopic techniques, which are in many cases specifically developed in the Department 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 NMR group under the direction of R. Kaptein 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 specific DNA sequences by proteins is a fundamental event, which lies at the root of e.g. the regulation of gene expression. 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. NMR spectroscopy is the method of choice for obtaining the experimental data that can be transformed into the desired three dimensional structures, because it is at present the only technique that in principle can give this information on biomolecules under natural physiological conditions.

At present the required detailed NMR spectroscopic information can only be obtained for relatively small biomolecules, up to a molecular mass of 20.000 Dalton, whereas 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. The group has been highly succesful in this area in its study of the lac-repressor DNA binding domain or "headpiece" and its interaction with a DNA "operator" fragment.
The figure shows a structure for this complex based on 2D NMR data. Similar studies are being done on lexA repressor, arc and mnt repressor and the receptors of the retinoic acid and glucocorticoid nuclear hormones, in order to establish the mechanism for the recognition of specific DNA sequences by proteins. At the moment it is already clear that several possible "structural motifs" are used in nature for specific recognition processes, but much has still to be learned about their detailed nature.

Methodology

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. One of the main research efforts of the group is therefore the development of new experimental techniques to alleviate this problem. The group has developed advanced (photo)chemically induced dynamic nuclear polarization techniques (photo-CIDNP) for the study of the surface structure of proteins and their interaction with ligands, thus providing information on the presence of certain amino acids in the active (binding) site of the protein.

Three- and four-dimensional NMR spectroscopy is another area that is being explored in order to alleviate the interpretation problem, and more specifically the ambiguity in the assignment of resonances. The succesful combination of various two-dimensional NMR techniques into non-selective three dimensional techniques has great promise for extending the molecular weight limits into the 30.000 Dalton area and furthermore, for the possible automation, at least partial, for the assignment procedures, one of the current bottlenecks in generating three-dimensional structures from NMR data.

Other important research topics involve the application of relaxation matrix methods in structure refinement, the use of NMR R-factors, the exploration and application (e.g. crambin) of direct NOE structure refinement methods, and automated secondary structure assignment.