Protein disorder is predicted to be widespread in eukaryotic proteomes, although direct experimental evidence is rather limited so far. To fill this gap and to unveil the identity of novel intrinsically disordered proteins (IDPs), proteomic methods that combine 2D electrophoresis with mass spectrometry have been developed. Here, we applied the method developed in our laboratory [Csizmo7acute;k et al., Mol. Cell. Proteomics 2006, 5, 265-273] to the proteome of Drosophila melanogaster. Protein Df31, earlier described as a histone chaperone involved in chromatin decondensation and stabilization, was among the IDPs identified. Despite some hints at the unusual structural behavior of Df31, this protein has not yet been structurally characterized. Here, we provide evidence by a variety of techniques such as CD, NMR, gel-filtration, limited proteolyzsis and bioinformatics that Df31 is intrinsically disordered along its entire length. Further, by chemical cross-linking, we provide evidence that it is a monomeric protein, and suggest that its function(s) may benefit from having an extended and highly flexible structural state. The potential functional advantages and the generality of protein disorder among chromatin organizing proteins are discussed in detail. Finally, we also would like to point out the utility of our 2DE/MS technique for discovering-or, as a matter of fact, rediscovering-IDPs even from the complicated proteome of an advanced eukaryote.
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