Structural characterization of Deinococcus radiodurans phytochrome in solution

Sammanfattning: Phytochromes are red/far-red photoreceptors which control a wide spectrum of biological processes inplants, fungi, and bacteria. Although phytochromes have been extensively studied, their mechanism ofaction still remains elusive. The signaling mechanism suggested by crystal structures involves refoldingof the so-called PHY tongue. However, currently, the involvement of the other two prominent structuralelements, the so-called helical spine and a knot in the peptide chain, remains unclear.Here, the first backbone assignment of the photosensory module of D. radiodurans phytochrome ispresented. To achieve a higher degree of the backbone assignment and thus gain further knowledge onthe conformational changes in the chromophore binding domain, an efficient method for refolding ofthe CBDPHY phytochrome fragment was developed. While further NMR measurements on therefolded phytochrome are needed, preliminary results already show the value of the extended backboneassignment for the future structural studies. Previously unreported changes in the knot region of thephotosensory module of D. radiodurans phytochrome were captured by solution NMR. All NMRobservables suggest photoinduced structural changes in the aforementioned region, implying that thesignal is carried from the chromophore to the helical spine, and through it, to the PHY domain and theoutput module. Furthermore, a study of the region near the bottom of the helical spine has beenconducted. Using several sets of the residual dipolar coupling (RDC) measurements, together with themolecular dynamics (MD) simulations, we gained a better understanding of the network of interactionsspanning from the chromophore to the dimer contact surface.Additionally, a procedure for structural analysis of nanoscale particles at XFELs using angular crosscorrelationsis outlined here. This approach was applied to the scattering data of ideal icosahedralparticles, a phytochrome and the human pre-ribosomal (pre-40S) particle. Condor was used to predictX-ray scattering amplitudes of these particles for customized experimental designs. Angularcorrelations can be accurately extracted from multiple-particle fluctuation X-ray scatteringexperiments. We concluded that angular cross-correlation functions (CCFs) preserve a substantialamount of structural information which enables the observation of structural features of particles at thenanometer scale. Consequently, correlation maps could be useful to follow fast dynamical changesin the structure, for instance, as a response to external stimuli.