Lidija Vrhovac: Structural characterization of Deinococcus radiodurans phytochrome in solution

Abstract

Phytochromes are red/far-red photoreceptors which control a wide spectrum of biological processes in plants, fungi, and bacteria. Although phytochromes have been extensively studied, their mechanism of action still remains elusive. The signaling mechanism suggested by crystal structures involves refolding of the so-called PHY tongue. However, currently, the involvement of the other two prominent structural elements, 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 is presented. To achieve a higher degree of the backbone assignment and thus gain further knowledge on the conformational changes in the chromophore binding domain, an efficient method for refolding of the CBDPHY phytochrome fragment was developed. While further NMR measurements on the refolded phytochrome are needed, preliminary results already show the value of the extended backbone assignment for the future structural studies. Previously unreported changes in the knot region of the photosensory module of D. radiodurans phytochrome were captured by solution NMR. All NMR observables suggest photoinduced structural changes in the aforementioned region, implying that the signal is carried from the chromophore to the helical spine, and through it, to the PHY domain and the output module. Furthermore, a study of the region near the bottom of the helical spine has been conducted. Using several sets of the residual dipolar coupling (RDC) measurements, together with the molecular dynamics (MD) simulations, we gained a better understanding of the network of interactions spanning from the chromophore to the dimer contact surface.

Additionally, a procedure for structural analysis of nanoscale particles at XFELs using angular crosscorrelations is outlined here. This approach was applied to the scattering data of ideal icosahedral particles, a phytochrome and the human pre-ribosomal (pre-40S) particle. Condor was used to predict X-ray scattering amplitudes of these particles for customized experimental designs. Angular correlations can be accurately extracted from multiple-particle fluctuation X-ray scattering experiments. We concluded that angular cross-correlation functions (CCFs) preserve a substantial amount of structural information which enables the observation of structural features of particles at the nanometer scale. Consequently, correlation maps could be useful to follow fast dynamical changes in the structure, for instance, as a response to external stimuli.