491:	Characterization of VCaB45, an Arabidopsis Calcium Binding Dehydrin.

Authors:	Torvik, Carl, F.(A)Randall, Stephen, K.(A)
Affiliations:	(A): Indiana University- Purdue Unversity, Indianapolis
Presenter:	Torvik, Carl F., cftorvik@iquest.net

We are characterizing VCaB45, a cold regulated Arabidopsis thaliana protein. We became interested in this protein because of its immunological cross-reactivity to a previously described celery vacuolar calcium binding protein (Celery VCaB45). An antibody specific to Celery VCaB45 was used to screen an Arabidopsis expression library and the recovered clones were sequenced. One of these clones showed 100% identity to a previously published mRNA encoding a type II LEA (the dehydrin ERD14). The protein encoded by this cDNA was expressed in E. coli using a vector which allowed high levels of protein expression following induction by IPTG. The resulting protein was shown to remain soluble when heated to 90oC in an aqueous solution. The protein was further purified by binding to an anion exchange column followed by elution with a NaCl gradient. Under our conditions, it eluted at a concentration of approx. 100 mM NaCl. Following purification, two assays were used to detect calcium binding: (1) A calcium overlay blot, which detects proteins that bind calcium through acidic amino acids or through structures capable of renaturing following SDS-PAGE, and (2) a filter binding assay, which measures 45Ca2+ associated with protein retained on a membrane. Both of these assays indicated that the protein bound calcium. We will be using equilibrium dialysis to further characterize the ion binding properties of this dehydrin, including a determination of the protein's Ka for calcium. The dehydrin was also shown to be phosphorylated by Casein Kinase II (and dephosphorylated by Shrimp Alkaline Phosphatase) in vitro. Investigations are in progress to determine whether the protein is phosphorylated in vivo and whether this phosphorylation has any effect on its affinity for calcium.