Abstract
While DNP has a history that goes back 60 years, high-field DNP is a young and rapidly developing field. Applications of DNP on proteins have been published from 1997 on, but stayed of limited use until a few years ago. The possibilities have not been fully explored. Two applications in
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the field of medicine and biomaterials have been presented in this thesis. Further, methodological developments using spin labels covalently attached to proteins are described. The thesis also contains the first data recorded at 800 MHz / 527 GHz system, its perfomance in terms of enhancement and resolution, and new insight in the structure of the membrane protein KcsA. The bacterial K+ channel KcsA has been investigated with the newly available 800 MHz/ 527 GHz DNP system. Comparison with 400 MHz / 263 GHz spectra, revealed a small line width improvement. Transverse paramagnetic relaxation only played a role for residues close to the protein surface. Residues more deeply buried in the membrane showed only minor or no PRE contribution to the line width. Comparison of line width and shape using MD simulations indicated that the majority of the linewidth increase can be explained by conformational heterogeneity of the residues in question. The freezing out of motion causes all conformations to be present in the spectra leading to major line broadening and therefore intensity loss. This is nicely demonstrated with residues T74 and T75 which were visible or broadened out in the spectra depending on the state in the gating cycle of KcsA. Quenching of the protein surface due to paramagnetic relaxation is not always prefered. By using site-directed labeling techniques, the location of the transverse paramagnetic relaxation can be controlled. Due to the tetrameric nature of the protein KcsA, the MTS spin labeling of a single cysteine mutation lead to four radicals close enough to facilitate the CE. We found a sizeable DNP enhancement for the KcsA mutant G116C (14.2 at 400 MHz) and were able to modulate the signals in the spectra by choosing a different labeling site of the protein KcsA. The liposomal Alzheimer’s vaccine Palm1-15 contains the N-terminal part of the protein Aβ. The peptide is retained on the lipid surface by palmitoyl anchors on both sides. Three amino acids were 15N13C labeled. Secondary chemical shifts extracted from 2D 2Q1Q correlations revealed more than one conformation for the peptide. The lipid composition of the liposome changed the chemical shift of the amino acids. MD simulations suggest more β-sheet-like oligomer formation of the peptide in the case of the lipid formulation DMPC/DMPG/Cholesterol compared to DMTAP/Cholesterol. Biominerals extracted from the marine unicellular eukaryote Stephanopyxis turris were investigated using DNP. Proteins protected from the SDS/EDTA treatment by the silica cell wall, were found. Also, resonances from polyamines were present in the NC-correlation spectra. No signs of protein-sugar links were found. The NMR data suggest that the secondary structure of the proteins contains a least a fraction of peptides that adopt a β-sheet conformation.
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