טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentDines Monica
SubjectStructure Determination of Proteins Involved in the
Stability of the Phycobilisomes during
Environmental Stress
DepartmentDepartment of Chemistry
Supervisor Professor Noam Adir
Full Thesis textFull thesis text - English Version


Abstract

The phycobilisome photosynthetic antenna complex, found in cyanobacteria and red-algae, interacts with proteins expressed specifically to deal with different forms of physiological stress. Under conditions of nutrient starvation, expression of the NblA protein is required in the process that leads to phycobilisome degradation and bleaching of the cells. HspA, a 16.5kDa heat shock protein expressed in cyanobacterial cells, has been shown to provide functional stability to the phycobilisome during heat stress. We have cloned the genes encoding for the NblA proteins from the thermophilic cyanobacteria Thermosynecococcus vulcanus (Tv-NblA) and from the mesophilic Synecococcus elongatus (Se-NblA) and for the HspA from T. vulcanus into bacterial expression vectors in order to determine their three-dimensional structures. The resulting recombinant proteins were found to be sparingly soluble, limiting their usefulness in the performance of crystallization experiments. We have developed a novel protocol that utilizes relatively high concentrations of urea to afford sufficient solubility to the protein. This has lead to the successful growth of diffraction quality crystals of these proteins. Complete data sets collected to 2-2.5Å from crystals of  proteins show that the crystals are stable, and useful for structure determination.

We report here the three-dimensional structure of the Tv-NblA to 2.5Å with and without urea. The monomer has a helix-hairpin-helix motif which dimerizes into an open four-helical bundle in the asymmetric unit, identical to the NblA dimer from Anabena, whose structure was previously determined. Comparison between structures and homology based models indicate that while the NblA family is only slightly homologous, there are certain attributes of the protein that remain constant, and could be involved in its function that is probably a structure-related function. We proposed here, also, a new theoretical model of interaction between the NblA and the phycobilisome that presents a new possibility of insertion of the NblA into the whole phycobilisome. 

The similarity between the structure of the Tv-NblA with and without urea shows that the presence of urea did not influence the protein structure, denaturation has not occurred and specific protein-protein interactions have been preserved. We believe that this protocol may be a generally advantageous method to obtain well diffracting crystals of sparingly soluble proteins.