טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentAilie Marx
SubjectCrystallization and Structure Determination of Phycobilisome
Components
DepartmentDepartment of Chemistry
Supervisor Full Professor Adir Noam
Full Thesis textFull thesis text - English Version


Abstract

Cyanobacteria and red algae possess unique antennae complexes called phycobilisomes.  A complete understanding of the mechanism by which the phycobilisome efficiently absorbs and transfers light to photosystem II requires the determination of an atomic level structure.  Aided by their abundance and solubility, numerous crystal structures of phycobiliproteins have been solved. The colourless, so called linker, proteins are in contrast relatively hydrophobic and unstable as an isolated component and to date only one crystal structure has been solved.  Lack of structural data is limiting functional understanding of linkers, thought to be involved in phycobilisome assembly and stabilization, and facilitation of efficiency and uni-directionality of energy flow down the complex.  This research aimed at improving the resolution of the phycobiliprotein phycocyanin, and crystallization and structure determination of the linker protein with which it is associated in vivo.


Fine tuning of crystallization and cryoprotection methods resulted in a diffraction pattern with a resolution higher than 1.3Å, an improvement on the highest reported resolution structure for any phycocyanin to date (PDB code: 1JBO; 1.45Å).  This structure along with three more phycocyanin structures determined in this project, each using a different cryoprotection condition,  were used for structural analysis of the functional hub, the cofactors.  Similarity of spatial arrangement, average B factors and the identification of water molecules consistently present at particular spatial positions were compared across the range of different structures and allowed for the identification of consistent, and so presumably functionally significant, features.   


Recombinant DNA technology was used to clone and sequence the gene encoding the cpcC rod linker from T. vulcanus.  Bioinformatic analysis of the primary sequence pre-empted difficulties encountered in obtaining a soluble solution of the cpcC protein.  Overexpression of cpcC in E. coli. yielded mainly inclusion bodies and various techniques were employed in pursuit of a more soluble protein solution.  Low concentration samples were set up for crystal screening however failed to provide single crystals.  In a final attempt to determine the linker structure, cocrystallization of the overexpressed linker with purified phycocyanin was attempted.  It was rationalized that the natural association of these two proteins and the high solubility and relative ease of crystallization of phycocyanin would provide a means of coercing the linker into a soluble, concentrated and crystallisable form.  Crystals were obtained which were visually similar to phycocyanin crystals and indeed their diffraction yielded structures identical to that of pure phycocyanin with no evidence of additional electron density.