NMR of solvent paramagnetic relaxation enhancements

NMR spectroscopy has developed into one of the principal methods in structural biology and enables not only the determination of the three-dimensional structure but also of the dynamic aspects of biological macromolecules. Relaxation enhancements upon the addition of an inert paramagnetic agent (solvent PREs) have been shown to be useful for determining the orientation and immersion depth of peptides bound to membrane-mimetics and also for the structure determination of proteins. Within this project we will extend the range of applications of relaxation enhancements by paramagnetic co-solvents and answer biological and biophysical questions about the role of a recently discovered, catalytically active, cation-p interaction in a transmembrane helix of yeast V-ATPase, dynamics of proteins as well as protein-ligand interactions. We will determine the requirements for keeping arginine in the hydrophobic environment of a membrane-mimetic and determine the pKa values of an arginine residue at different positions of a transmembrane helix. Answering these questions will help in unraveling the molecular details of the function of catalytically active arginine residues in a series of membrane-bound proteins. Solvent paramagnetic relaxation enhancements (PREs) will also be used for probing the dynamical behavior of soluble proteins through a comparison of experimental with theoretical PREs. Conformations which are invisible in regular NOE-based NMR structures or in an X-ray structure still influence the PRE, which contains information about the solvent accessibility of a certain nucleus. This approach will be employed on proteins which are partially or completely intrinsically unstructured to identify regions containing lowly populated structured states. Finally, transferred PREs will be used to determine the orientation and insertion depth of small molecule ligands bound to large protein receptors. Transferred PREs do not rely on chemical modification or isotopic labeling and can be applied to any kind of proteins without a size limitation. The information obtained can be used as experimental input in ligand-protein docking calculations or to identify the part of a drug candidate where chemical modification has the highest potential of changing the binding strength.

NMR spectroscopy is probably the most often used technique for the structural characterization of organic and biomolecules. Interatomic distances are the main structural restraints, which can be extracted from NMR spectra. Within this project we have developed new strategies to extract structurally relevant information by using inert paramagnetic compounds in the solvent. These paramagnetic co-solvents increase the linewidths of NMR spectra in a distance-dependent way. Since the whole solvent is made paramagnetic, changes in the signal linewidth provide information about the distance of a certain atom to the molecular surface. This information was used for example to elucidate the catalytic mechanism of the enzyme V-ATPase, which plays an important role in the regulation of the pH value, so the concentration of hydrogen ions within the cell. Malfunction of this enzyme is for example involved in the development of osteoporosis. By using paramagnetic line-broadening we could show that the interaction of two specific amino acids is responsible for the transport of hydrogen ions by this enzyme. Another application was the investigation of the interaction of macrolide antibiotics with bile acids by paramagnetic co-solvents. Together with a group from the University of Zagreb we could show that binding of these antibiotics to bile acids does not reduce their activity and that different macrolide antibiotics all show the same binding mechanism to bile. Paramagnetic co-solvents have also been employed for a more exact determination of protein structures, which should increase their resolution as needed for structure-based drug design.