טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentSuissa Szlejf Maayan
SubjectStrructural and Functional Characterization of Terminal
Subunits of Cyanbacterial Phycobilisomes
DepartmentDepartment of Chemistry
Supervisor Professor Noam Adir
Full Thesis textFull thesis text - English Version


Abstract

The major light harvesting antenna in cyanobacteria is the phycobilisome (PBS). The PBS typically contains different subunits, and energy efficiently flows from high (phycocyanin, PC) to low (allophycocyanin, APC) energy absorbing subunits. PC and APC are both composed of two basic subunits - α and β, which assemble into monomers (αβ), trimers (αβ)3, hexamers and rods. The smallest PBS identified to date is that of Acaryochloris marina (A. marina), composed of a single rod. The crystal structure of A. marina phycocyanin (AmPC), the major component of the A. marina PBS, was previously determined to be a heterodimer of two alpha and two beta isoforms, superimposed in the asymmetric unit of the PC monomer structure. Several AmPC types were isolated, among them a PC type exhibiting allophycocyanin (APC)-like characteristics, suggesting it can efficiently serve as the PBS terminal emitter, since no such component was identified for the A. marina. The lack of APC along with the novel PC types, suggest that the A. marina PBS may operate without APC, solely based on one multi-functional PC. Here we focus on structure determination of this APC-like PC. In comparison, a mutant strain of S. elongatus PCC 7942 (olive), which lacks PC rods in its PBS due to an induced mutation, relies only on core cylinders containing APC in order to sustain itself. Since the inner arrangement of the subunits and linkers in the cylinder is apparently important to the organism and is yet unclear, we focused on isolation of the core from the mutant cyanobacteria in order to determine its structure and significance in the photosynthetic process.
Experimentally, each isolation protocol was optimized according to the proteins’ biochemical properties. For the core complex, a purified protein solution was obtained that resulted in the growth of crystals, but no diffraction images could be collected after X-ray irradiation of the crystals. However, the solution was taken to an electron microscope where an image was obtained showing the cylindrical structure of the core. In addition, a model of the olive core, built based on a previously solved PBS structure, predicts the position of the ApcE subunit in the complex which may serve as a connecting chain between neighboring core complexes within the cell. We suggest further experiments with cryoEM to try and solve the structure of the core and to better understand the properties of this chain. For the A. marina APC-like subunit (TPC), large crystals were obtained, for which X-ray diffraction images were collected. The structure is yet to be solved, though confocal microscope measurements show energy emission at ~670nm, which support the hypothesis of TPC being the terminal emitter subunit in the A. marina PBS. We suggest further processing of the data in order to solve the structure. In addition, we suggest further refining of the isolation protocol and crystallization conditions in order to better purify the protein, as well as further characterizing it using methods such as mass spectrometry, to reveal the effect of the isoform composition on the energy transfer.