Font Size: a A A

Investigation Of The Assembly Mechanism And Functional Regulation Of Phycobilisomes-the Light-Harvesting Complex Of Cyanobacterial Photosynthesis

Posted on:2014-02-11Degree:DoctorType:Dissertation
Country:ChinaCandidate:N ZhangFull Text:PDF
GTID:1220330398959962Subject:Microbiology
Abstract/Summary:PDF Full Text Request
Phycobilisomes (PBSs) are the dominant light-harvesting multi-protein complex in cyanobacteria and red alage, composed of phycobilin-attached phycobiliproteins (PBPs) and colorless linker polypeptides, absorbing solar light and transferring energy to the reaction center in the thylakoid membrane. Different PBPs are assembled into PBSs with the aid of distinct linkers. So far, the investigations on PBPs have been detailedly carried out. However, the progress about linker polypeptides is much slower. To date, only a crystal structure of cyanobacterial core linker polypeptide has been reported. The linkers play an important role in the linkage and location of PBPs and the adjustment of energy transfer pathway. Thus, it is necessary to study the interaction between PBPs and linkers for better understanding of the structure and function of PBSs. In addition, PBSs function as a key factor in energy regulation. Rapid long-range PBSs diffusion has been reported as a prerequisite for light-state transitions. However, the electron microscope data have implied a diverse arrangement of cyanobacterial thylakoid membranes which is not homogeneous and continuous in some cyanobacterial strains. Furthermore, the vertical space between parallel thylakoid membrane layers in most cyanobacteria is too narrow for PBS diffusion, given the size of most single cyanobacterial PBS. The spatial hinderance would limit PBS diffusion in a long-range. Therefore, it is still questionable whether or not the PBSs could diffuse rapidly on the thylakoid membrane, and needs further extensive and deep investigation on cyanobacterial PBS fluorescence dynamics, which will be advantageous for our better understanding on energy regulation and dissipation mechanism. Thus, focusing on the structure and function of PBSs, we carried out systematic research from different aspects, and have made some progresses as follows: 1. FRAP investigation on two unicellular cyanobacteriaFluorescence recovery after photobleaching (FRAP) technique is applied to track the distribution, diffusion and other dynamic behaviors of various molecules labed with a fluorescent tag in living cells. Here, a detailed investigation was carried out in two unicellular cyanobacterial strains with different PBP compositions, Synechococcus sp. WH7805(WH7805) and Synechococcus sp. PCC7002(P7002). There are two ubiquitous large cyanobacteria groups that are categorized by PBP composition:the allophycocyanin (APC)+phycocyanin (PC)+phycoerythrin (PE) type (PE-type) and the APC+PC type (PC-type). According to previous reports, P7002is a PC-type cyanobacterium, and WH7805is categorized as a PE-type. Based on PBP compositions in two cyanobacterial strains, lasers of555nm and639nm were used to excite the PEs and PCs at the distal end of their PBS rods, respectively. Fluorescence recovery was observed in the native cells of both PC-type and PE-type cyanobacterial strains, which was consistent with previous reports on PC-type cyanobacteria. Furthermore, we used treatment with glutaraldehyde, a very effective protein cross-linker, to fix PBSs firmly upon the surface of thylakoid membranes. However, fluorescence recovery also occurred in glutaraldehyde-fixed cells, a little lower than that in native cells under similar bleach depth. In addition, we isolated PBSs and PBS-thylakoid membranes from two strains, and analyzed the integrity of these two kinds of samples by spectral methods. The fluorescence also recovered in isolated and glutaraldehyde-fixed PBSs and PBS-thylakoid membranes after photobleaching. Mean fluorescence recovery percentages were calculated in FRAP of two kinds of cells before and after glutaraldehyde treatment. Quantitative analyses indicated no significant differences in the fluorescence recovery levels between the native and glutaraldehyde-fixed cells for one cyanobacterial strain by Unpaired Student’s t-test (P<0.05). These results suggested that fluorescence recovery following photobleaching in cyanobacteria could not result from the long-range lateral diffusion of PBSs on the thylakoid membrane surface. Because all fluorescent molecules can undergo reversible photobleaching, cyanobacterial fluorescence recoveries in two unicellular strains as measured by FRAP could be an intrinsic in situ photoprocess of bleached PBSs rather than long-range PBS diffusion.2. FRAP investigation on two filamentous cyanobacteriaTo further find out the popularity of fluorescence recovery attributed to intrinsic photoprocess, we chose two filamentous cyanobacteria from distinctive environments to carry out similar FRAP investigations. Pseudanabaena sp.0831(P0831) was isolated from from Arctic sea ice, and Pseudanabaena sp. FACHB-1277(F1277) was grown in freshwater environment. As there were no reports about PBP compositions of these two strains, first it is necessary to perform PBP composition analysis. With absorbance spectral analysis, two filamentous cyanobacteria were defined as one PC-type strain (P0831) and one PE-type strain (F1277), respectively, compared with absorbance spectra of WH7805and P7002. Based on the compositions of PBPs, lasers of555nm and639nm were chosen to excite PE at the distal of PBS in F1277and PC at the distal of PBS in P0831. Partial fluorescence recovery was observed in both native cells. Furthermore, fluorescence recovery also occurred in glutaraldehyde-fixed cells, a little lower than that in native cells under similar bleach depth. Moreover, the fluorescence also recovered in isolated and glutaraldehyde-fixed PBSs and PBS-thylakoid membranes after photobleaching. Quantitative analyses indicated that there were no significant differences in the fluorescence recovery levels between the native and glutaraldehyde-fixed cells for one cyanobacterial strain, with a combination of the calculation of mean fluorescence recovery percentage and Unpaired Student’s t-test (P<0.05). These results suggested that fluorescence recovery following photobleaching in cyanobacteria could not result from the long-range lateral diffusion of PBSs on the thylakoid membrane surface. Therefore, quantilative and quantitative investigations both in vivo and in vitro reveal that fluorescence recoveries in two filamentous cyanobacteria as measured by FRAP could be an intrinsic in situ photoprocess of bleached PBSs, as it is in unicellular cyanobacteria.3. Insights into the assembly mechanism of the phycobilisome rod in cyanobacteriaPfam00427domain, as the most widely-spread and conservative domain in cyanobacterial linker polypeptides, is rarely investigated. The crystal structure of pfam00427domain has been analyzed by Dr. Gao Xiang in our lab. Based on the crystal structure of pfam00427domain, we revealed the interaction style between pfam00427and C-PC, and proposed a precise model for the architecture of the PBS rod by biochemical and simulation studies.Here, we simulated the pfam00427-C-PC (αβ)6complex without any artificial residue restrictions by using the AutoDock program. A total of50solutions were recorded, and only the top14solutions were collected for further analysis because the binding energy of the subsequent results rose up rapidly, which implied that these results were against biophysics rules. Generally, the14solutions were divided into two groups according to the relative positions of pfam00427in the C-PC (αβ)6cavity, with models from the same group matching each other well spatially with a slight30°rotation. To find out that which group is close to native state, further biochemical verification was needed. To better elucidate the interaction mode of pfam00427and C-PC, a modified GST pull-down assay was applied to identify the potential interaction residues on pfam00427. A dozen of key amino acid residues of pfam00427domain involving the interaction with C-PC were point-mutated. The regular GST pull-down assay was modified. A mild denaturation and renaturation process was adopted after the mixture step. To verify the feasibility of the GST pull-down assay, C-PC alone or with GST protein was loaded on the resin after the same treatment and analyzed by SDS-PAGE. The results eliminated the possibilities of a nonspecific binding of C-PC to the resin and an interaction between the GST protein and C-PC. Additionally, both GST-pfam00427and C-PC showed no detectable conformational or spectrographic changes after this weak denaturation and renaturation process. In this assay, the wild-type pfam00427exhibited obvious interaction only with the C-PC β-subunit, while the band of the C-PC a-subunit was almost missing on the SDS-PAGE gel. Simultaneously, seven mutants (R26E, E29R, E31R, D33R, R41E, H48A and R54E) were observed to significantly reduce or even abolish the pfam00427-C-PC interaction. All of the seven identified residues were gathered at a relatively conserved surface patch of pfam00427that harbors plenty of charged residues, designated as "C-PC-binding patch". We compared the models of Group One and Group Two and found that only models from Group One can satisfy these limitations. We further chose the candidate bearing the largest buried surface area to a single β-subunit chain from Group One as our initial model to run a molecular dynamics simulation for structural optimization. Based on these biochemical results, we further chose a reasonable candidate model to run a molecular dynamics simulation for structural optimization, and obtained the final resulting pfam00427-C-PC (αβ)6heptamer model.Based on our pfam00427-C-PC (αβ)6heptamer structural model and the previous APC-LC7.8crystal structure, we proposed a precise model for cyanobacterial PBS rod. First, we generated a structural model for LRT (pfam01383) by homology modeling using Lc7.8as a template. We therefore docked LRT into the central hole of the opposite C-PC (αβ)3according to the APC-Lc7.8crystal structure. As the domain organization in the different linkers is repetitive from distal to proximal to the core, this packing mode is applied to the whole PBS rod. In our assembly model, the LR spans and connects two adjacent C-PC (αβ)6hexamer with its linking region (-60residues) being located between two C-PC (αβ)6hexamer and accessible to the solvent. The PBS rod is terminated by an LRT at the distal end and is joined to the PBS core through an LRC at the proximal end. Our final simulated model for the molecular structure of PBS rod reveals the mechanism that how PBS rod assembles and elongates clearly, which also modified the PBS rod model previously proposed.4. The crystallization and optimization of pfam00502domain of Lcm in cyanobacterial phycobilisomeAs a composition of the trimeric (αβ)2(LCMβ18) terminal energy acceptor, Lcm is the most important linker protein in the PBS core complex, and transfers energy from PBS to reaction center. The N-terminal domain of LCM, pfam00502, is a PBP-like domain. The underlying mechanisms of the auto-chromophorylation of pfam00502and the detailed structure of TEA have been elaborated. However, how the pfam00502domain achieves the auto-chromophorylation and participate in the assembly of PBS core complex and PS Ⅱ is far from known, due to the lack of structural information of pfam00502. In this chapter, we tried to analyze the crystal structures of apo-pfam00502and PCB-pfam00502, in order to dig out their differences and explain the auto-chromophorylation process.First, apo-pfam00502from model cyanobacteria Synechocystis sp. PCC6803was expressed and purified. Preliminary crystal screening was performed at4℃and18℃, respectively. However, apo-pfam00502crystallization was difficult, as the apo-pfam00502was conformationally unstable due to the lack of PCB. In preliminary crystal screening, we finally obtained a protein crystal verified by X-ray diffraction. However, the crystal was too small for data collection, and needs further optimization. In addition, PCB-pfam00502was expressed by in-vitro recombination and purified. Preliminary crystal screening was conducted at18℃. Nevertheless, no PCB-pfam00502crystal was observed so far through many trials, and it may be ascribed to the heterogeneity of protein samples which resulted from the incomplete PCB-catalysis in vitro. In addition, whether or not the PCB-pfam00502needs further chemical modification was unknown, which may have an effect on PCB-pfam00502crytallization.
Keywords/Search Tags:Cyanobacteria, Phycobilisome, Linker Polypeptides, Pfam00427domain, Pfam00502, Fluorescence Recovery after Photobleaching
PDF Full Text Request
Related items