One key to two doors Dual targeting peptides and membrane mimetics
Sammanfattning: A targeting peptide at the N-terminus of a precursor protein usually directs the protein synthesized in the cytosol to a specific organelle in the cell. Interestingly, some targeting peptides, so-called dual targeting peptides (dTPs) can target their protein to both mitochondria and chloroplasts. In order to understand the mechanism of dual targeting, a dTP from threonyl tRNA synthetase (ThrRS-dTP) was investigated as a model dTP in this thesis work. The results suggest that ThrRS-dTP is intrinsically disordered in solution but has an ?-helical propensity at the N-terminal part. Tom20 and Toc34 are the two primary receptors on the outer membranes of mitochondria and chloroplasts, respectively. We found that the N-terminal half of the ThrRS-dTP sequence, including an amphiphilic helix, is important for the interaction with Tom20. This part also contains a ????? motif, where ? represents a hydrophobic/aromatic residue and ? represents any amino acid residue. In contrast, neither the amphiphilic helix nor ????? motif in ThrRS-dTP has any special role for its interaction with Toc34. Instead, the entire sequence of ThrRS-dTP is important for Toc34 interaction, including the C-terminal part which is barely affected by Tom20 interaction.In addition, the role of lipids in the organelle membrane for the recognition of dual targeting peptides during protein import is also the focus of this thesis. The tendency to form ?-helix in ThrRS-dTP, which is not observable in solution by CD, becomes obvious in the presence of lipids and DPC micelles. To be able to study such interactions, DMPC/DHPC isotropic bicelles under different conditions have also been characterized. These results demonstrate that bicelles with a long-chained/short-chained lipid ratio q = 0.5 and a concentration larger than 75 mM should be used to ensure that the classic bicelle morphology persists. Moreover, we developed a novel membrane mimetic system containing the galactolipids, MGDG or DGDG, which have been proposed to be important for protein import into chloroplasts. Up to 30% MGDG or DGDG lipids were able to be integrated into bicelles. The local dynamics of the galactolipids in bicelles displays two types of behavior: the sugar head-group and the glycerol part are rigid, and the acyl chains are flexible.
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