CRYSTAL STRUCTURE OF THE PLANT FULL-LENGTH SSDNA BINDING PROTEIN STWHY2 IN FREE

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The plant defense response to pathogens relies primarily on the use of pre-existing physical barriers and inducible defenses, which require massive transcription of defense genes. The whirly protein family was discovered when studying the activation of such defense gene. StWhy1 was found as the activator of the potato plant gene PR-10a that is activated during defense response by binding to the promoter in its single stranded conformation. The protein was later crystallized and revealed novel tetramer whirligig-like¿ folding. As we tried to move forward in identifying the mechanism used by the members of this gene family, we have encountered difficulties taking the genetic approach. The phenotype shown by various Knockout of those genes are rare and subtle. Since we suspect the C-terminal part of those proteins to play big role in the Whirly function, we would like to obtain structural data of this region. Since the StWhy1 construct used then was not full length and it had a tag in place of C-terminal end, we need to use new construction. A paralogue of StWhy1, StWhy2, show a great potential for crystallization and hope to use this characteristic to obtain good crystals of the free form and the form in complex with ssDNA. We hope that those constructions will help reveal the location and potential role of the C-terminal domain when the protein is free or bound to DNA. This knowledge will guide our design of new experiments in the lab and lead us to better understanding of this protein family that plays an important role in plant defense against pathogens.