A3.1: Caged puromycin

This PhD student will focus on the uncaging mechanism of ortho-nitrobenzyl (oNB) type protecting groups and the optimization of these compounds for puromycin uncaging. Based on the Wachtveitl group’s recent studies on a photolabile puromycin synthesized by the group of Schwalbe with 4,5-dimethoxy-2-nitrobenzylchloroformiat (NVOC) as protecting group, one of the tasks of this PhD student is to work on improved cages. He/She will thus perform kinetic experiments to determine the timescale of puromycin photorelease by time resolved spectroscopy and IR-detection of photo-intermediates and -products, monitor the release kinetics of the nascent polypeptide chain and compare the results to in vitro folding data. He/She will test the performance of newly developed caging groups in time-resolved experiments (synthesis will be performed in PhD thesis B1.1). Specific tasks of this thesis are:

  • Perform fs-time resolved transient spectroscopy on improved photocages.
  • Comparison of the measured data with theory.
  • Setup and characterization of a laser synchronization used for a gapless time-resolved detection of uncaging dynamics from the fs to the ms time scale.
  • Test the applicability of the novel photolabile groups for the kinetic investigation of posttranslational folding event.

 

A3.2: Two-photon and wavelength-selective uncaging

With ultrafast vibrational and electronic spectroscopy this PhD student will follow the reaction pathways of wavelength-selective and two-photon uncaging in oligonucleotides and peptides as specified in the proposal for PhD theses A2.1 and A2.2 (organic synthesis) and the quantum chemistry part of PhD thesis A1.2 (and with the advice by the associated research group of Dreuw) the PhD student will obtain a detailed picture of the uncaging kinetics.
The PhD student will also work on novel types of two-photon cages consisting of a sensitizer (e.g. an efficient two-photon dye) / cage combination. Photoactivatable peptides developed in PhD thesis A2.2 and studied in PhD thesis B4.3 will be characterized and tested as two-photon cages. Furthermore the PhD student will try to establish 3D-clustering of biomolecules by two-photon excitation.
In addition to the two-photon cages the PhD student will study the photocleavage of spectrally shifted cages. Color tuning of the photolabile group allows constructing multiply caged compounds that can be converted to the active molecule in a sequential fashion. This will be a key technique for the central aim of this RTG to develop complex uncaging scenarios, indispensable for the synchronized observation of multi-step reaction sequences.

 

A3.3: Kinetic separation of folding and association dynamics triggered by differentially caged ATP

This PhD thesis will be a mechanistic and kinetic study on larger biological systems and thus represents a direct application of the principle of wavelength selective, sequential uncaging described above. The PhD student will learn and use different time-resolved techniques including time correlated single photon counting and rapid scan FTIR spectroscopy to obtain a complete picture of the sequential processes of helicase binding and RNA unwinding that are expected to occur on a nested hierarchy of time scales.
In close collaboration with PhD thesis B2.1 the PhD student will perform photochemical and kinetic experiments on differentially caged ATP molecules. The site-specific introduction of a photo-removable roadblock into the target RNA is a prerequisite to kinetically disentangle association and propagation of the helicase. After photoinduced removal of the road-block, the propagation of RNA unfolding can be spectroscopically probed either by FTIR or by time-resolved fluorescence spectroscopy. In combination with the NMR studies performed in PhD thesis B2.1, this integrated approach should lead to a detailed molecular picture of the complex reaction sequence of RNA unwinding.
The second part of this thesis is also focused on the functional dynamics triggered by the release of ATP. The PhD student will be responsible for the spectroscopic characterization of response of diacylglycerol kinase incorporated into lipid cubic phase (LCP) upon ATP uncaging developed in PhD thesis B3.1.