NMR structure determination of biomolecules

The applied research program is aimed at improving current NMR methodology and contributing with advanced experimental methodology to structural aspects of important molecular biology problems. It will focus on research projects related to such important biological topics as "Cell growth and Cell differentiation - Mechanism of protein assembly and protein recognition". Specifically, it is planned: Refinement of the solution structure of cysteine-and glycine-rich protein CRP2 using uniform 12C, 15N-labelling. The structural studies will provide an important starting point of experimentation for identifying the physiological partners and the basis for structure elucidation of complexes with its physiologically relevant binding partners. Structural analysis of point mutants of CRP2 will provide a detailed molecular picture about the structural determinants of the global fold and the thermodynamic stability of CRP2. NMR studies at higher concentration will reveal information about the molecular details of CIRP2 self-assembly, which might serve as a model for CRP2`s protein recognition capabilities, a key molecular event by which CRP2 plays an important role in the regulation of gene expression. Paramagnetic derivatives of CIRP2 will provide important information about the electronic details of zinc- coordination and the influence of additional stabilizing interactions (e.g. hydrogen bonding). This information should be of importance to protein engineering and the construction of novel metal binding proteins. Structural and spectroscopic information about the apo-form of CRP2 will be extremely beneficial for solution NMR structural studies of ligated CRP2 once the physiological targets of CRP2 have been identified. Intended areas of NMR methods development are: It is planned to quantify auto- and cross-correlation relaxation mechanism of protein backbone nuclei to define protein long-range order and demonstrate its applicability to NMR protein structure determination. These schemes should provide important additional structural and dynamical information and should improve structure determination entirely based on NOEs. To achieve a more accurate description of protein backbone dynamics, it is intended to combine the merits of a recently developed heteronuclear relaxation experiment comprising adiabatic fast passage with the improved resolution of triple- resonance correlation schemes. Additionally, new experiments originally designed for backbone dihedral angle determination will be modified to determine protein side-chain dihedral angles.