Research goals

• We will focus on two systems catalyzing the production of cyclic guanylate monophosphate (cGMP) and cyclic adenylate mononucleotide (cAMP) that serve as second messengers in diverse signalling processes in various eukaryotes including mammals. The strategic concept includes combination of different light sensor modules with various catalytic domains using genetic engineering to tailor the sensor-catalyst communication and the catalytic performance.

• We will express and purify resulting fusion enzymes, and study their catalytic activity as a function of illumination. Different fusion variants will be screened to identify enzymes with sufficient levels of activity and light regulation.

• The design of these systems will be accompanied and supported by detailed spectroscopic studies covering a wide dynamic range. The spectroscopic investigations will allow probing femto- and picosecond events of the light-absorbing cofactor by transient absorption and stimulated Raman spectroscopy and cofactor-protein relaxation processes from the nanosecond to second time-scale by time-resolved RR and IR spectroscopy.

• Of utmost importance, however, is the elucidation of the coupling between protein structural changes of the photoreceptor and the conformational transition of the catalytic module which causes the activation of the enzyme. We will introduce spectroscopic reporter groups into the proteins preferentially in the contact domain between sensor and catalyst. Specifically, EPR spin-label spectroscopy and FRET techniques will be employed to monitor the molecular basis of the signal transduction from the sensor to the catalytic module.

• In addition, we will attempt to crystallise these constructs for determination of their three-dimensional structures. The performance of the designed catalysts will be tested in hippocampal neurons and in hippocampal rat slices and in Drosophila neurons, respectively, as a prerequisite for optogenetic applications.