Studies of structural dynamics of photoactive proteins in solution using time-resolved X-ray scattering

Abstract

Photoactive proteins that exhibit unique three-dimensional structural transitions in response to light convert light signals into chemical signals using their structural properties and regulate various biological reactions in living organisms. Therefore, to understand the biological reactions in living organisms, it is important to investigate the structural dynamics of photoactive proteins. In this regard, the structural changes in the photoreactions of the circular oligomer photoactive yellow protein, bacteriophytochrome, and PixD were investigated using the time-resolved X-ray solution scattering technique. Based on the light-induced N-terminal protrusion of photoactive yellow protein, novel protein-based photoswitches, circular oligomer photoactive yellow proteins, were designed and generated. In the design of the photoswitches, the N-terminus of each photoactive yellow protein unit is connected to the C-terminus of the other unit by a linker. From the design, it was expected that the circular oligomer photoactive yellow proteins would exhibit expansion motion. However, time-resolved X-ray solution scattering study show that the circular oligomer photoactive yellow proteins exhibit light-induced structural changes that gradually contract. The result suggest an approach to generate a novel protein-based circular oligomer photoswitch. In addition, it shows the need to elucidate the structural properties of the photoswitches and suggests a method to investigate the structural properties. The time-resolved X-ray solution scattering also applied to phytochrome, which exhibit unique structural dynamics in response to red light in living organisms. Phytochromes are categorized into a canonical form and a bathy form. Based on crystallography studies on phytochromes, it was expected that the photoreactions of canonical and bathy phytochromes would include different three-dimensional structural changes, respectively. However, information on the light-induced structural dynamics of bathy phytochromes is still elusive. Therefore, the difference between the structural properties of canonical and bathy forms remains an open question. In this study, the time-resolved X-ray solution scattering reveals that bathy phytochromes exhibit light-induced structural changes in terms of an “O” shaped geometry in the central helical backbones. This result suggest that a structural mechanism distinct from canonical phytochromes, which exhibits a “Y”-shaped geometry in the central helical backbones, is related to the photoreaction of bathy phytochromes. In nature, proteins form protein complexes using protein-protein interactions. The dissociation of the protein complexes plays an important role in various biological reactions such as energy metabolism and cell signaling. Therefore, investigating the structural dynamics of dissociation of protein complexes can provide important information for understanding various biological mechanisms. In this regard, time-resolved X-ray solution scattering were applied to the PixD, a photoactive protein that dissociates from one decamer to five dimers in response to light. This X-ray scattering study revealed structural information involved in the dissociation of the PixD. From the structural information on the dissociation of PixD, it is suggested that the dissociation of protein complexes occurs as changes initiated in the local structure of the complex propagate to the overall structure.