19F PCS NMR spectroscopy: a novel approach to determine structures of protein-ligand complexes, illustrated with human carbonic anhydrase II and fluorinated inhibitors

This thesis presents the structural investigation of human carbonic anhydrase II (hCA-II) inhibitor complexes by pseudocontact shift (PCS) NMR spectroscopy. Five different single cystein mutants of hCA-II were prepared in different isotope labelling schemes. These protein mutants were tagged with LnM8-SPy complexes for the determination of PCS. Triple labelling of one protein mutant allowed the unambiguous assignment of 90 % of the protein backbone. For four out of five protein mutants reasonable PCS were observed and the assignment of the PCS was possible for more than 90 % of the assigned residues. This large number of assigned PCS showed an excellent agreement between the protein structure in solution and the X-ray structure. In order to determine precise ∆χ-tensor parameters based on the X-ray structure PCS in the neighbourhood of the tag and in fluctional parts of the protein were systematically excluded. 19F PCS were determined for two different fluorinated inhibitors bound to hCA-II, for each of the four protein mutants. Based on the determined ∆χ-tensor parameters the fluorine position was determined based on the 19F PCS alone. This is the first example of the localization of a fluorine-containing ligand within a protein, based only on one-dimensional 19F-nuclear magnetic resonance (NMR) spectra and the PCS derived from this data.
While the fluorine-fluorine distance in the ligand that contains two F-atoms, was reproduced very precisely by the 19F-PCS, the positioning of the two ligands within the hCA-II protein was significantly different compared to the X-ray structures of the two complexes. For one of the two protein inhibitor complexes, a deviation of up to 8 Å was observed. The most likely reason for this systematic error was identified to be residual anisotropic chemical shift (RACS) that could not be taken in account in the structure calculation. This was supported by the second protein-inhibitor complex, where a higher degree of motional freedom of the fluorine atoms and therefore smaller RACS were observed and consequently the determined PCS position was closer to the X-ray structure. A validation of the PCS structure determination suggested a precision of the proton position from PCS alone of 1-2 Å. Finally, the principle of using an 1H-19F-heteronuclear Overhauser effect spectroscopy (HOESY) spectra to determine H PCS of protons close to the fluorine was demonstrated.