טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentDavid Liron
SubjectThe Phycobilisome Complex
DepartmentDepartment of Chemistry
Supervisor Professor Noam Adir
Full Thesis textFull thesis text - English Version


Abstract


The Phycobilisome (PBS) is the major light harvesting antenna in cyanobacteria and red algae. The PBS is a gigantic complex which is made up of pigmented (phycobiliproteins) and unpigmented proteins (linker proteins). Our goal is obtain an atomic resolution structure of the entire PBS complex from the cyanobacterium T.vulcanus and A. marina that has the simplest of all PBS by combining x-ray crystallography with electron microscopy.

The crystal structure of PC-Rod was determined to a resolution of 1.5 Å with a monomer in the asymmetric unit. Within the rod cavity, very weak and unstructured electron density contributed by linkers could be identified. MS and confocal microscopy showed that the crystals contained functional rods. Based on our results a new model for PC rod was suggested which indicated that the main role of the linkers is to modify the spectral properties of the phycobiliproteins.

T.vulcanus PBS crystals were obtained and analyzed by fluorescence, SDS-PAGE and mass spectrometry (MS). The results indicate that the crystals contain intact, functional PBS complex. The crystals diffracted to about 3.5Å and we found that the PBS is arranged in the crystal as elongated rods. This organization is different from the suggested PBS model based on electron microscopy but still transfers energy from PC to APC. PBSs underwent mild chemical cross-linking and thus intact and functional PBSs could be isolated in low salt conditions that exhibited similar spectral properties as the non cross-linked PBS. New crystals of the cross-linked PBS diffracted to about 3.5Å and seemed to have similar organization as the non cross-linked PBS. Negatively stained TEM micrographs of PBS in high phosphate indicated that T. vulcanus PBS is constructed from a pentacylinderal core and at least six rods with dimensions of 48 x36 nm. Cryo-TEM initial model for cross-linked PBS presented a spherical particle with estimated dimensions of 24x24 nm. Into this particle four rods of four hexamers can be fitted. This model may support our current conclusion that probably the complex is being arranged in the crystal as elongated rods.

Small crystals of A. marina PBS were obtained. Although the crystals seemed to be homogenous and functional according to our emission measurements from a single crystal, the crystals didn’t diffract.We determined the molecular weight of A. marina PBS before and after crystallization by Fluorescence Correlation Spectroscopy measurement that indicated that molecular weight was ~1.4MDa as was expected from an intact A. marina PBS.