RESUMO
PYP-phytochrome related (Ppr) protein contains the two light sensor domains, photoactive yellow protein (PYP) and bacteriophytochrome (Bph), which mainly absorb blue and red light by the chromophores of p-coumaric acid (pCA) and biliverdin (BV), respectively. As a result, Ppr has the ability to photoactivate both domains together or separately. We investigated the photoreaction of each photosensor domain under different light irradiation conditions and clarified the inter-dependency between these domains. Within the first 10 s of blue light illumination, Ppr (Holo-Holo-Ppr) accompanied by both pCA and BV demonstrated spectrum changes reflecting PYPL accumulation, which can also be observed in Ppr containing only pCA (Holo-Apo-Ppr), and a fragment of Ppr lacking the C-terminal Bph domain. Although Holo-Apo-Ppr showed PYPL as a major photoproduct under blue light, as seen in the Bph-truncated Ppr, the equilibrium in the Holo-Holo-Ppr was shifted from PYPL to PYPM as the reaction progresses under blue light. Concomitantly, the spectrum of Bph exhibited subtle but distinguishable alteration. Together with the fact, it can be proposed that Bph with BV influences the photoreaction of PYP in Ppr, and vice versa. SAXS measurements revealed substantial tertiary structure changes in Holo-Holo-Ppr under continuous blue light irradiation within the first 5 min time domain. Interestingly, the changes in tertiary structure were partially suppressed by photoactivation of the Bph domain. These observations indicate that the photoreactions of the PYP and Bph domains are coupled with each other, and that the interplay realizes the structural switch, which might be involved in downstream signal transduction.
RESUMO
Self-assembled protein nanostructures have gained interest, owing to their potential applications in biomaterials; however, successful design and construction of protein nanostructures are limited. Herein, we constructed fusion protein 1 by linking the C-terminus of a dimerization domain and the N-terminus of another dimerization domain with a three-helix bundle protein, where it self-assembled mainly into tetramers. By replacing the C-terminal dimerization domain of 1 with a trimerization domain (fusion protein 2), hexamers were mainly obtained. According to ab initio structural models reconstructed from the small-angle X-ray scattering data, the tetramer of 1 and hexamer of 2 adopted quadrangle and cage-like structures, respectively, although they were combinations of different conformations. High-speed atomic force microscopy observations indicated that the tetramer and hexamer exhibit conformational dynamics. These results show that the present method utilizing three-helix bundle-linked fusion proteins is useful in the construction of protein nanostructures.
