|Ph.D Student||Salama Faris|
|Subject||Engineering a Photosystem II Dependet Bio-generator|
and the X-Ray Structure of HspA, which Protects
PSII from Thermal Damage
|Department||Department of Chemistry||Supervisor||Professor Noam Adir|
|Full Thesis text|
In an age of ever progressive depletion of resources and environmental concerns, we have sought to develop a means to harness the natural process of energy production used in photosynthetic organisms. The initial steps of oxygenic photosynthetic electron transfer occur within Photosystem II. We have identified a site, poised in the vicinity of the QA intermediate acceptor, which could serve as a potential electron transfer conduit to redox active proteins. Here we show that modification of Lysine 238 of the D1 protein to glutamic acid (Glu), in Synechocystis sp. PCC 6803, results in a strain that grows photautotrophically while its isolated membranes are able to perform light-dependent reduction of cytochrome c with water as the electron donor. Cytochrome c photoreduction by the Glu mutant also protects the D1 protein from photodamage. This new pathway may serve as the first component of a biotechnology-based energy production system.
Photosystem II, the first protein complex in the photosynthetic chain, found in plant chloroplasts, algae and cyanobacteria is the most stress sensitive component in the chain. HspA, a small, 16.5 kDa, Heat shock protein expressed in cyanobacteria Thermosynecococcus vulcanus (T. vulcanus), has been showed to provide a functional stability to Photosystem II during heat stress.
Working with isolated photosynthetic membranes can lead quickly to damage caused by heat stress. In order to mimic the thermal stability process that occurs in cyanobacteria, we overexpressed the HspA protein from two cyanobacterial species, using a bacterial expression vector, in order to determine its 3D structure and characterize its biochemical properties. The resulting recombinant proteins were found to be sparingly soluble, limiting their usefulness in the performance of crystallization experiments. Using a method, that was developed in our laboratory; we solubilized the protein at concentrations of urea high enough to afford sufficient solubility to the protein. We successfully crystallized the protein and obtained diffraction to 2.3Å resolution from the HspA from T. vulcanus. Surprisingly, the solution of the structure revealed a protein in the early stage of denaturation that caused by the presence of the urea molecules. The new structure completely lacks its secondary structures however its 3D conformation appears to have remained intact.