Study On The Active Conformations Of Phycobiliproteins From Cyanobacteria And Red Algae

Phycobilisomes (PBSs) are the major light harvesting pigment-protein complexes in cyanobacteria and red algae, and they are composed of various phycobiliproteins (PBPs) and linker polypeptides. Based on spectra properties, phycobiliproteins are commonly divided into three main groups:allophycocyanin (APC), phycocyanin (PC) and phycoerythrin (PE). PBPs are covalently linked with several co-factors named phycobilins, which enable PBPs to harvest solar energy and transfer it.PBSs in cyanobacteria and red algae locate on the surface of thylakoid membrane, directly exposing to cytoplasm or plastid stroma. PBPs may be directly affected by changes in cytoplasm or plastid stroma. For the PBPs in cyanobacteria and red algae, it is crucial to maintain their light-harvesting and energy transfer ability with respect to certain conformational changes. Crystal structures of PBPs provide us with static information. For a better understanding of the protein conformation, crystallography information of the proteins of interest is required to be combined with active structural and functional investigations.This thesis discussed the active conformations of the PBPs from cyanobacteria and red algae. The main results are as follows.1. Efficient separation and purification of APC from Spirulina platens isAPC is the core component of a PBS. It plays important physiological role in cyanobacteria and red algae, and it is also widely used in biochemical techniques and food industry. Here we established a method for extracting APC from Spirulina platensis with high efficiency. After pretreating the crude extract, we used hydroxylapatite to enrich and extract APC from PBP solution, and large amount of APC could be separated from other PBPs. This extracting method could not only separate large amount of APC in short time, but also obtain high purity of APC. APC extracted from crude extract could be further purified with single step of ion-exchange chromatography to obtain APC with higher purity. 2. Research on the active conformation of the APC from Spirulina platensisWe used the APC purified from Spirulina platensis, and studied its spectra property variations in response to pH changes. Light-harvesting ability was monitored by absorption spectra. Energy transfer ability was monitored by fluorescence emission spectra. Secondary structure was monitored by circular dichroism (CD) spectra. At the meantime, we studied the aggregation state of APC by absorption spectra, fluorescence spectra and vis-CD spectra. By analysis of spectra properties, we found that APC showed good absorbance and fluorescence stability at varying pH, with only minor changes between pH 4-10. The trimeric structure of APC was maintained while local variations of protein peptides were also shown in response to the environmental disturbance. Beyond this pH range, secondary structure as well as overall conformation of APC dramatically changed, and the energy absorption and transfer ability were also disrupted. By analysis of APC crystal structure, we found some key amino acid residues on the interaction surfaces betweenαandβsubunits. The fundamental tertiary of APC structure was probably stabilized by these contacts contributed by specific protein residues, whereas there might be some local conformational variations in response to environmental changes. Thus the physiological stability of APC was maintained.3. Research on the active conformation of the C-phycocyanin from Spirulina platensisWe purified the C-phycocyanin (C-PC) from Spirulina platensis. Variations of solution pH were used to disturb C-PC structure, and dynamics of structural and functional changes of C-PC were monitored in response to different pH values. We studied the light-harvesting ability (revealed by absorption spectra) and energy transfer ability (revealed by fluorescence spectra). At the mean time we studied changes of secondary structure (revealed by CD spectra) and aggregation state (revealed by vis-CD spectra). By analysis of these results, we found that C-PC could maintain its aggregation state even when there were some disturbances in solution, but the secondary structure might be flexible in a certain distance. Subsequently we analyzed crystal structures of C-PC, especially the interaction surfaces which maintain the aggregation state of the protein. We found some key sites on the interaction surfaces. By the interaction of these key amino acid residues, the aggregation state of C-PC was stabilized. Thus it is suggested that when certain environmental disturbance happens, these key sites can stabilize the aggregation state of C-PC, but in other regions the conformation of polypeptide could be flexible in response to environmental changes. Thus, C-PC maintained its functional stability with respect to certain conformational variations.4. Investigations of active conformational variation and the pH sensitivity of R-phycoerythrin from Polysiphonia urceolataPE is the characteristic PBP in red algae. We purified R-phycoerythrin (R-PE) from Polysiphonia urceolata and studied its active conformational variations and pH sensitivity. We found that, between the range of pH 3.5-10, the conformation of chromophores in R-PE was relatively stable, which was revealed by the stable absorption and fluorescence spectra properties of R-PE. While in this pH range, there were some variations in the amounts of secondary structures, the major structure is always a-helix. No P-sheet structure was detected. Amounts ofβ-turn and random coil structure varied in small scale. In acidic environment lower than pH 3.25, conformations of aromatic amino acid residues were exposed to the solution. In basic solution with pH higher than 10, there were conformational changes as well as deprotonation process of the chromophore. Fluorescence properties of R-PE disappeared in extreme pH. When pH was lower than 3.5 or higher than 10, amounts of a-helix decreased dramatically, while amounts ofβ-sheet and random coil structures increased. We analyzed the dynamics of spectra properties in response to solution pH, and found that the energy transfer ability in the protein was not influenced. We also found that extreme pH could not only induce denaturation of R-PE but also modulate spectra properties of the chromophores although the way of modulating was different between acidic and basic environment. Therefore, R-PE could maintain its light-harvesting and energy transfer ability in spite of some conformational variations. Then we analyzed the crystal structure of R-PE. Similar to other PBPs, there were some amino acid residues with strong interactions on the interaction surfaces of R-PE subunits. With these interactions, R-PE maintained its aggregation state. When environment conditions changed, the key regions, which are responsible for functional performances, maintained the structures and stabilized the energy absorption and transfer abilities of R-PE, while other regions showed some flexibility in response to disturbance.5. Interaction of aromatic amino acids with chromophores in PBPsAromatic amino acids are rich in PBPs. Fluorescence from aromatic amino acids changes following the folding/unfolding process of proteins. We monitored pH induced unfolding process of APC by fluorescence from aromatic amino acids. At the meantime, we found that when aromatic amino acids were excited, fluorescence from phycobilins arose in visible region of the spectra, suggesting there is energy transfer pathway from aromatic amino acid residues to the chromophores. We also found that when phycobilins were excited indirectly, fluorescence from APC was different from that when phycobilins were excited directly. By comparison of indirectly excited fluorescence from different PBPs and analysis of phycobilin conformations, we suggest that there are two types of phycocyanobilins in APC, which can emit fluorescence at 660 nm and 639 nm respectively. The existence of 639 nm fluorescence and other questions are also discussed.