B1.1: Light-induced protein nascent chain folding
- Preparation of so-called stalled ribosomes in which the polypeptide nascent chain is still attached to the ribosome. The protein and RNA biochemistry has been established.
- Synthesis of caged puromycin. A first generation photoprotecting group (with 4,5-dimethoxy-2-nitrobenzylchloroformiate (e(NVOC) group) could already be synthesized. New photocaging groups will be developed based on coumarin systems. For optimization of the chemistry, help from the Heckel group will be provided.
- Characterization of the photophysics together with the group of Wachtveitl.
- Study of protein folding of staphylococcal nuclease released from stalled ribosomes by time-resolved NMR triggerd by uncaging puromycin from the photoprotective precursor.
B1.2: Complex RNA folding reactions populating various intermediates
- Two RNA sequences will be prepared containing orthogonal photo-protecting groups. One sequence is a 78mer RNA able to adopt either a conformation exhibiting cleavage reactivity or a second conformation with ligase activity. Incorporation of photo-protecting groups at selected sites (orange symbols) will force the sequence to adopt the ligase conformation. Additionally, a second RNA molecule that serves as a substrate for the ligase reaction will be added.
- Light NMR experiments will be conducted, utilizing two laser coupled to the same NMR spectrometer to release the substrate strand and folding and chemical reaction in the ligase conformation can proceed.
- Subsequently, the ligated system is deliberated from the ligase fold by a second laser pulse. Conformational switching can occur to the equilibrium state of cleavage-ribozyme and ligase fold and the cleavage reaction can be monitored.
B1.5: Conformational dynamics of DNA G-quadruplexes and i-motifs investigated by time-resolved NMR spectroscopy
Beside double-helix conformations, oligonucleotides can also form non-canonical structures including tetraplex structures formed by pairing of four strands. These tetraplex structures include G-rich G-quadrupluexes and C-rich i-motifs. These structures can act as regulatory units in cells. The project B1.3 will utilize the concept of conformational caging to characterize the kinetics of folding of these DNA regulatory units, following previous work in the group.
The work will involve the synthesis of caged oligonucleotide strands in the Heckel group, the ligation of caged and uncaged strands in the Schwalbe group and the time-resolved characterization of folding reactions by NMR spectroscopy.
B1.6: Conformational dynamics of RNA ribozymes investigated by cage chemistry, chemical footprinting and time-resolved NMR spectroscopy
RNA molecules are unique in their ability to adopt multiple long-lived conformations with almost identical stability but high energetic separation. This can lead the population of multiple states for a single conformation that can exhibit distinctly different biological function. The investigation of their interconversion is particularly interesting. The project B1.4 will utilize the concept of conformational caging to characterize the kinetics of folding of bistable RNA structures.
A second aim of the project is to develop methods for the time-resolved footprinting of RNA conformation. Close collaboration with the Heckel and the Wachtveitl group are foreseen.
B1.7: Design and chemical synthesis of new caged metabolites
Riboswitches represent an important class of RNA regulation elements. Upon of metobolites, ligands of low molecular weight, the expression of genes that are involved in the metabolism of the cognate ligand, are up- or downregulated. But to now, 25 different classes of riboswitches have been reported. In previous work, we have developed a caged version of one specific metabolite.
The project B1.5 will develop new caging strategy, ultimately to develop a chemical biology tool box of light regulatable small molecules.
Close collaboration with the Heckel and the Wachtveitl group are foreseen.