Paramagnetic tools for the structural analysis of high molecular weight proteins

Paramagnetic effects provide important structural and dynamic information of biomolecules. However, the attachment of lanthanides through small chelating organic molecules to proteins (the most common way to obtain paramagnetic effects) requires single cysteine mutants, optimization of the tagging reaction and previous knowledge of the three-dimensional structure of the target to select proper attachment sites. In this work was developed a new method that relieves most of these disadvantages: the lanthanide is not directly attached to the target protein, but instead to a "reporter" protein that binds and transmits paramagnetic information to the target protein. In this thesis is shown that the attachment of a lanthanide in different locations on the surface of the reporter protein PDZ allows measuring residual dipolar couplings and pseudo contact shifts from several independent molecular alignments on any target. This is shown for ubiquitin and the maltose binding protein. The fusion of a 7-residue PDZ recognition peptide to the C-terminus of the target proteins is the only necessary modification to obtain the paramagnetic restraints. Therefore, this method allows recording a large amount of paramagnetic information from orientationally independent molecular alignments in proteins. Moreover, it is not necessary to have previous knowledge of the three-dimensional structure of the targets