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, the NblA protein is required for the process that leads to phycobilisome degradation and bleaching of the cells. HspA, a 16.5 kDa 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 these proteins 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 2-2.5 Å from crystals of both proteins shows that the crystals are stable, and useful for structure determination. A preliminary structure of the NblA shows that 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.
Bibliographical noteFunding Information:
We gratefully thank the staff of the European Synchrotron Radiation Facility for provision of synchrotron radiation facilities and assistance (beamlines ID29 and ID14). We would like to thank Dr. Orly Dym and the Israel Structural Proteomics Center for assistance in HspA crystallization trials. We thank Fares Salame for technical help. This work was supported by the Israel Science Foundation founded by the Israel Academy of Sciences and Humanities (438/02 and 683/03-17.1 to N.A. and R.S. respectively) and the German Israel Foundation (I-729-27.9/2002 to R.S.).
- Heat shock