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.