Design, syntheses and performance evaluation of novel generations of lanthanide chelating tags and an NMR study of formylglycine-generating enzyme

In order to provide cutting-edge tools for probing structure and dynamics of proteins by the use of lanthanide chelating tags (LCTs), new generations of LCTs with novel structural features and enhanced properties were developed. First, the presence of multiple conformational species and the averaging of paramagnetic effects by motional flexibility of the LCT on the surface of the protein was addressed. Subsequently, the development of an activator moiety that leads to a short, reduction-stable linkage formed upon tagging of the protein yielded an LCT with unprecedented induced anisotropy parameters and favorable ligation kinetics. Furthermore, the design and synthesis of a novel LCT that is suitable for tagging of synthetic RNA strands containing a modified nucleobase extension was achieved. Thereby, the repertoire of paramagnetic nuclear magnetic resonance (NMR) spectroscopy using LCTs was extended from the protein to the ribonucleic acid (RNA) world. Additionally, a comprehensive study about the formylglycine-generating enzyme (FGE) with its unusual fold and catalytic properties and the investigation of four novel antigens in cancer research was undertaken. An abstract of the five parts of this thesis is given in the following.
In the first part of this thesis, improved generations of LCTs are included. In order to push the limits in paramagnetic NMR spectroscopy using LCTs, one existing and three completely novel 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-derived LCTs were extensively studied. The exploration of the field started with the synthesis and performance evaluation of the eight-fold methyl-substituted Ln-DOTA-M8-(4R4S)-SSPy (DOTA-M8-(4R4S)- SSPy: (2R,2'R,2''R)-2,2',2''-((2S,5S,8S,11S)-2,5,8,11-tetramethyl-10-((R)-1-oxo-1-(2-(pyridin- 2-yldisulfanyl)ethylamino)propan-2-yl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)tripropano- ate) that is locked in only one conformational species. Subsequently, an LCT with four isopropyl substituents in the basic ring scaffold, i.e. Ln-P4M4-DOTA, was designed and synthesized (P4M4-DOTA: (2R,2'R,2''R)-2,2',2''-((2S,5S,8S,11S)-2,5,8,11-tetraisopropyl-10- ((R)-1-oxo-1-(2-(pyridin-2-yldisulfanyl)-ethylamino)propan-2-yl)-1,4,7,10-tetraazacyclodode- cane-1,4,7-triyl)tripropanoate). Both the Tm- and Dy- complex of the novel LCT displayed strongly enhanced pseudocontact shifts (PCSs) on both ubiquitin and hCA II constructs when compared to Ln-DOTA-M8-(4R4S)-SSPy. The isopropyl-substitution pattern was then combined with a reduction-stable linker suitable for in cell applications (P4T-DOTA: (2R,2'R,2''R)-2,2',2''-((2S,5S,8S,11S)-2,5,8,11-tetraiso-propyl-10-((2-(methylsulphonyl)thia- zolo[5,4-b]pyridine-5-yl)methyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)tripropionate). Due to the combination of the isopropyl substituents on the basic ring scaffold and the short linker moiety, the obtained PCSs and residual dipolar couplings (RDCs) were further increased when compared to Ln-DOTA-M8-(4R4S)-SSPy and Ln-P4M4-DOTA. The different stages of development then culminated in the rational design of Ln-M7-Nitro (M7-Nitro: Ln-(2R,2'R,2''R)- 2,2',2''-((2S,5S,8S,11S)-2,5,8,11-tetramethyl-10-((6-(methylsulphonyl)-5-nitropyridin-2-yl)- methyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)tripropanoate), a novel LCT exhibiting unprecedented anisotropy parameters on ubiquitin S57C, a reduction-stable linker, and strongly improved ligation kinetics, i.e. tagging of the cysteine residue of the investigated protein constructs to an extent greater than 95% over 30 minutes under close to physiological conditions (highly diluted (90 μM), aqueous buffer, pH 7.0). The favorable properties of the Ln- M7-Nitro LCT are mainly based on its novel, highly reactive and selective 5-nitro-substituted 6-(methylsulphonyl)-2-methylpyridine activator moiety.
The second part of the thesis comprises a review about the design and applications of LCTs for the investigation of proteins, nucleic acids, carbohydrate and complexes that was published in Progress in Nuclear Magnetic Resonance Spectroscopy. Subsequently, a review about the application of LCTs for the localization of ligands within biomacromolecules is included that will contribute as book chapter to Comprehensive Coordination Chemistry III.
The third part of this thesis deals with the transfer of the rich LCT toolbox from proteins to RNA. RNA molecules become increasingly important for future drug design and there is currently no LCT available that enables a successful covalent conjugation to RNA strands and subsequent exploitation of the paramagnetic effects induced by the lanthanide ions for structure elucidation. Therefore, an LCT comprising a novel activator moiety was designed and benchmarked on a 36-nt RNA strand that incorporates a modified nucleobase. The detected anisotropic effects with the newly designed LCT correspond within a range of 10% to those obtained with Ln-DOTA-M8-(4R4S)-SSPy on an ubiquitin S57C construct and it can therefore be concluded that the PCS methodology was successfully transferred from the protein to the RNA world and will contribute to future studies of structure, dynamics and ligand-binding of RNA molecules in solution.
In the fourth part of the thesis, a solution NMR study of structure, dynamics and mechanism of the FGE protein is included. The study yields residue-specific insights into the solution state structure and dynamics of a uniformly 2H, 13C, 15N labeled FGE construct and a uniformly 15N labeled FGE construct by use of amide proton exchange, T1 and T2 relaxation and heteronuclear Overhauser effect NMR experiments. Furthermore, the catalytic site of the enzyme was investigated by following chemical shift perturbations (CSPs) in 1H,15N-HSQC experiments recorded with samples of the apoenzyme, Cu(I)- and Ag(I)-containing enzyme as well as with several mutations and with and without substrate loading. Subsequently, the CSPs in 1H,15N-HSQC experiments recorded on a disease-related mutant of FGE were analyzed in order to provide a molecular basis for the occurrence of specific phenotypes in patients with multiple sulfatase deficiency (MSD). The solution NMR study extends the current knowledge about this highly special and exciting copper-containing enzyme.
The fifth and last part of this thesis comprises a study about a novel class of antigens in cancer research. Four novel antigens were synthesized and structurally characterized by NMR and HRMS. The biological experiments showed that the antigens stimulate T cells and can therefore be exploited for activation of specific T lymphocytes in order to recognize cancer cells. This novel class of antigens offers thereby an enormous therapeutic potential for the development of a potential vaccine against different types of cancer.