Spectroscopic investigation of phytochromes and NIR fluorescent variants
In this work, vibrational spectroscopic methods (resonance Raman [RR] and infrared absorption [IR]) were used for the investigation of phytochromes. Phytochromes are bistable photoreceptor proteins that bind bilins (BV, PCB, PΦB) as chromophores. Various plant, cyanobacterial, and bacterial variants regulate biological processes through the light dependent switching between metastable states that absorb red and far-red light (Pr and Pfr). The aim of the work was the elucidation of the mechanisms of photoinduced processes in different phytochromes. In the first part of the work, plant phytochromes were investigated. As in many (cyano-)bacterial variants, the Pr and Pfr states of plant phytochromes are not homogeneous, but instead exist as conformational equilibria of sub-states (I, II). As shown in this work, the distribution between Pfr-I and Pfr-II in plant phytochromes is affected i.a. by the N-terminal extension and binding of PIF proteins (phytochrome interaction factors), which in turn modulates the lifetime of the Pfr state. Furthermore, the photosensor constructs with and without the PHY domain (PAS-GAF-PHY [PGP], PAS-GAF [PG]) were analyzed in solution and in the crystalline state. While the respective Pr states have unaltered chromophore structures, PCB adopts a configuration similar to the Meta-Rc state in the photoproduct of the truncated PG variant. The investigations on cyanobacterial phytochromes in the second part of the work focused on the PCB-binding cyanobacteriochrome (CBCR) Slr-GAF3, which converts to a metastable green-absorbing (Pg) state after photoactivation of Pr. The unusual green absorption properties were analyzed on the basis of four different models proposed for the Pg state. A plausible explanation for the hypsochromic shift of the absorption bands is a twisted configuration of the pyrrole rings A and D in combination with a hydrated chromophore binding pocket. At low temperatures, Slr-GAF3 converts to an orange absorbing (Po) state. Various possible mechanisms of this transformation were discussed. The work on bacterial phytochromes (Bphs) in the third part of the dissertation comprises the decoding of thermal reaction mechanisms in the photocycle. For this purpose, a setup for time-resolved measurements was developed, which combines a flow-through system with a commercial FT-Raman spectrometer. Special features of the design are the continuous excitation in combination with a pre-resonant NIR measuring laser and the photochemical back-conversion of the sample in a reservoir. This is necessary to ensure that the dark-adapted state is the starting point for repetitive experiments. Using this technique, it was shown that the decay of the meta-states of the Bphs Agp1 and Agp2 not only leads to the formation of the active state, but also in an unproductive thermal shunt reaction back to the respective dark-adapted state. In the studies on the phytochrome-activated di-guanylyl cyclase IsPadC, the IR spectra suggest a reduced structural change of the PHY domain, which is essential for the formation of the activated (Pfr) state. These results confirm a Meta/Pfr heterodimer model, which was developed on the basis of time-resolved UV/vis data. In the fourth part of the thesis, the structural factors that influence the ratio of the different decay processes of the electronically excited state (Pr *) were investigated in order to allow the rational design of NIR-fluorescent Bphs (iRFPs). The Pr state of Bphs comprises a mixture of fluorescent (Pr-I) and non-fluorescent (Pr-II) substates that can be distinguished by RR spectroscopy. Therefore, the generation of highly fluorescent variants for fluorescence microscopy requires the shift of the conformational distribution towards Pr-I, as shown for iRFP713. The conformational equilibrium is affected by the substitution of amino acids both in the direct environment of the chromophore and at remote positions. Some iRFP variants with very high fluorescence quantum yields (FQY) contain a second cysteine in the GAF domain, which can also form a thioether bond to BV. Here, about 50% of the total protein ensemble contains the double-bound BV. It was shown in this work that the resulting internal crosslinking leads to a strong increase of FQY (16.6%) in iRFP682. RR spectroscopy indicates that the fluorescence gain, analogous to iRFP713, is due to an equilibrium shift towards Pr-I.
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