טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentTal Ofir
SubjectInvestigation of the Interactions Leading to Phycobilisome
Assembly
DepartmentDepartment of Chemistry
Supervisor Professor Noam Adir
Full Thesis textFull thesis text - English Version


Abstract

The Phycobilisome (PBS) of Thermosynechococcus vulcanus is an extremely large light harvesting complex, composed of the chromophore-carrying Phycocyanin (PC) and Allophycocyanin (APC) subunits and additional linker proteins. Models of the general architecture of the PBS have been suggested, based on electron microscopy low-resolution images. Despite the large number of crystal structures of most of the PBS components, a critical question regarding the interaction and energy flow between PC and APC is still unresolved. Additionally, the arrangement of the PBS components located inside the core cylinders is unknown.

We isolated functional T. vulcanus PBS, and performed cross-linking just sufficiently to isolate specific inter-subunit interaction units. Using MS analysis, we were able to identify cross-linked peptide pairs that were structurally close in the isolated complex. Independently of the cross-link set, we identified thousands of potential docking interactions of the different protein pairs. When we assessed the compatibility of each model with the cross-link set, we found specific interactions within the PBS sub-components that enable us to suggest possible functional interactions between the chromophores of the rods and core, and to improve our understanding of the assembly, structure and function of PBS. Additional characterization methods, including X-ray crystallography, Electron microscopy and spectroscopies, were utilized to assess functional aspects of the identified interfaces.

Model of the core and model of rod-core interaction were obtained and a unique pH-triggered biochemical mechanism was proposed for the assembly and disassembly of rods and core. The interacting amino acids in the proposed mechanism were found as evolutionary conserved residues. Using virtual mutations scanning, we found the same interacting residues as critical in the rod-core interaction.

The energy transfer from PC to APC was suggested to be via the PC(β84) - APC(α84) chromophores pair. Based on the proposed model of rod-core interaction, we conclude that the parallel-rods PBS model successfully describes the complex, avoiding the disarrangement of rods and core that presence in other proposed models. 

The inner arrangement of the core was illustrated, showing that the ApcE N-terminal domain's-loop might have important structural role in attaching the core cylinders together in the right way to enable energy transfer to the photosystems via the minor core components. We also suggested an arrangement of the linker proteins inside the rods cylinders, and a full rod-assembly model. The rods and core linkers, based on the results, have a functional role in energy-transfer tuning in the PBS by possible interactions with the surrounding chromophores.