The Design Of Tetrapyrrole Photoreceptor And Dynamic Structure Of Trptophan Photoreceptor

On photosynthesis, the capability to perceive and respond to light signal are essential for cynobacteria and plants to survive and adapt to the ever-changing environments on earth. All these vital movements are dependent on photoreceptor. The light-harvesting photoreceptors take charge of capturing solar energy for photosynthesis, which provide the energy for all living organism. The signaling receptors are responsible for perceiving and responding the light signals, then regulating the growth and development. In this paper, we mainly studied the light-harvesting photoreceptors (PBS) in cynobacteria and the UV-B signaling photoreceptor (UVR8) in Arabidopsis thaliana.The phycobilisomes of cynobacteria and red algae are highly efficient peripheral light-harvesting complexes that capture and transfer light energy in a cascade of excitation energy transfer steps through multiple phycobilin chromophores to the chlorophylls of photosystem centers. In this work, we focus on the last step of these processes by constructing simple functional analogues of natural phycobilisomes that are based on bichromophoric protein complexes comprising a phycobilin-and a chlorophyll-or porphyrin-binding domain. The former is based on ApcE(1-240), the N-terminal chromophore-binding domain of the phycobilisomes’core-membrane linker(LcM), and the latter on HP7, a de novo designed four-helix bundle protein that was originally planned as a high-affinity heme-binding protein, analogous to b type cytochromes. We fused a modified HP7protein sequence to ApcEA, a water-soluble fragment of ApcE(1-240) obtained by excising a putative hydrophobic loop sequence of residues77-153. HP7was fused either to the N-or the C-terminus of ApcEA or inserted between residues76and78, thereby replacing the native hydrophobic loop domain. We describe the assembly, spectral characteristics, and intramolecular excitation energy transfer of two unique systems:in the first, the short-wavelength absorbing zinc-mesoporphyrin is bound to the HP7domain and serves as an excitation-energy donor to the long-wavelength absorbing phycocyanobilin bound to the ApcE domain; in the second, the short-wavelength absorbing phycoerythrobilin is bound to the ApcE domain and serves as an excitation energy donor to the long-wavelength absorbing zinc-bacteriochlorophyllide bound to the HP7domain. All the systems that were constructed and tested exhibited significant intramolecular fluorescence resonance energy transfer with yields ranging from21%to50%. This confirms that our modular, covalent approach for studying EET between the cyclic and open chain tetrapyrroles is reasonable, and may be extended to larger structures mimicking light-harvesting in cyanobacteria. The design, construction, and characterization process demonstrated many of the advances in constructing such model systems, particularly in our ability to control the fold and aggregation state of protein-based systems. At the same time, it underlines the potential of exploiting the versatility and flexibility of protein-based systems in assembling multiple pigments into effective light-harvesting arrays and tuning the spectral properties of multichromophore systems.Arabidopsis thaliana photoreceptor UVR8(AtUVR8) undergoes dimer dissociation upon absorbing UV-B light. Monomeric UVR8directly interacts with key regulators in plant light signaling pathways thereby modulating cellular processes involved in UV protection and circadian rhythms. UVR8is unique among all known photoreceptors:it does not require cofactors to capture a UV photon. However, the molecular mechanism of UV-B perception and structural events that lead to dimer dissociation remain elusive. Based on the static crystal structure analysis and mutational studies, we found the hydrogen bond between D129-W233and the two salt bridges between R286-D107and R338-D44at the dimer interface are essential for stabilizing the dimer structure. And the clustered tryptophan residues at there are responsible for UV-B responses in AtUVR8. Analyzing the conformational and spatial distributions of13Trp residues in the AtUVR8structure, we propose that Trp residues act as UV-B pigments and constitute a light-harvesting antenna similar to light harvesting complexes in cyanobacteria and plants. And through the study of absorption and fluorescence characteristic, and the Trp fluorescence lifetime of the proteins, we proved that these antenna pigments collect broad band UV-B irradiation, funnel excitation energy to the epicenter W285-W2333and triggers a UVB-induced structural signal. To gain mechanistic understanding of UV-B signaling, we further apply dynamic crystallography to directly probe transient structural changes associated with primary photo-events in AtUVR8. We found the early structural events involve localized and concerted torsional motions in Trp285and Trp233, and the disappearance of a highly ordered water molecule at the tight-knit dimer interface of AtUVR8. Finally, integrated all these results, we proposed the UVR8photosensitive and signaling mechanism. All these studies provide a theoretical basis for developing and using UV-B photoreceptors.In addition, we preliminarily studied the T type lyase (CpcT) in Nostoc.sp.PCC7120and the new blue light photoreceptor (OsHAL3) in Oiyza sativa. Now we mainly introduced the crystallization of selenomethionine-substituted CpcT and OsHAL3, and the crystal of CpcT-PCB has been obtained by soaking apo-CpcT crystals with PCB. The resolution of the crystals CpcT, CpcT-PCB and OsHAL3are1.9A,2.5A and2.9A, respectively. Through analyzing and comparing the structure of CpcT and CpcT-PCB, we found the key structure which associated with catalytic mechanism of this lyase. It was provided a structural basis for further catalytic mechanism study. Though analyzing the structure of OsHAL3and comparing with the structure of AtHAL3, we proposed a hypothesis about the photochemistry and photo-induced mechanism of OsHAL3.It was indicates the direction for further physiology study.