| Description |
For many decades, it was assumed that protein function was dependent on the ability of polypeptides to fold into stable three-dimensional structures specified by their amino acid sequences. But since about 2000, it has become apparent that there are functional proteins, or portions of proteins, that are highly disordered in their functional state. Members of this class of proteins, known as intrinsically disordered proteins (IDPs), do not form unique folded structures in isolation, though portions of many fold upon binding other molecules. It is estimated that approximately 30% of eukaryotic proteins contain at least one disordered region greater than 50 residues. IDPs have been found to participate in a large number of cellular processes, including transcriptional regulation and signal transduction. In many cases, they appear to act as flexible linkers between more rigid structures. Late embryogenesis abundant (LEA) proteins are a large family of IDPs that are expressed at high levels during the later stages of plant seed development. Dehydrins are subfamily of LEA proteins that are believed to play a role in protecting cellular contents from the effects of dehydration. The goal of this project was to study the physical traits of XERO2, a 193 amino acid residue LEA protein from the model plant Arabidopsis thaliana. BL21 E. coli cells were transformed with a synthetic XERO2 gene, and the expressed protein was isolated using a new purification process that included chromatography on phosphocellulose. This method yielded ~30 mg of protein per liter of bacterial culture with a purity of ~90%. The average hydrodynamic radius (RH) of XERO2 was estimated using size exclusion chromatography (SEC) and calibration with well-characterized globular proteins. In phosphate buffer at pH 7.2, RH was determined to be approximately 32 Å, a value that is intermediate between those expected for a 193-residue fully unfolded polypeptide (42 Å) and a globular protein of the same size (22 Å). Comparisons with other IDPs suggest that the degree of expansion of XERO2 lies well within the observed range for this class of proteins. Preliminary small-angle X-ray experiments also indicate that XERO2 is extensively disordered and has a significant tendency to aggregate at concentrations of about 10 mg/mL. |
