Intrinsic Structural Disorder of DF31, a Drosophila Protein of Chromatin Decondensation and Remodeling Activities Edit Szo ˝llo ˝si, † Monika Bokor, ‡ Andrea Bodor, § Andras Perczel, § Eva Klement, | Katalin F. Medzihradszky, |,⊥ Kalman Tompa, ‡ and Peter Tompa* ,† Institute of Enzymology, Hungarian Academy of Sciences, Budapest, Hungary, Research Institute for Solid State Physics and Optics of Hungarian Academy of Sciences, Budapest, Hungary, Laboratory of Structural Chemistry and Biology, Institute of Chemistry, Eo ¨tvo ¨s Lora ´nd University, Budapest, Hungary, Proteomics Research Group, Biological Research Center, Hungarian Academy of Sciences, Szeged, Hungary, and Department of Pharmaceutical Chemistry, University of California San Francisco, San Francisco, California 94143 Received November 8, 2007 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 [Csizmo ´ 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 discoveringsor, as a matter of fact, rediscoveringsIDPs even from the complicated proteome of an advanced eukaryote. Keywords: intrinsically unstructured protein • natively unfolded protein • 2D electrophoresis • chemical cross-linking • differential scanning calorimetry Introduction Intrinsically disordered proteins (IDPs) carry out their func- tions despite their lack of well-defined 3D structures. 1–4 Often, these proteins function by molecular recognition, in which structural disorder confers specific advantages, such as rapid and specific binding, the capacity of binding multiple partners, or binding promiscuity, that is, the ability to bind different partners with distinct functional outcomes. 5 Currently, we have solid experimental evidence for the disorder of about 500 proteins, 6 but bioinformatics predictors developed to recognize protein disorder from amino acid sequence suggest that several thousand IDPs may exist in the human proteome alone. 7,8 The extension of the structure-function paradigm to encompass IDPs requires the detailed structural-functional characteriza- tion of many novel IDPs, in order to arrive at useful generaliza- tions in terms of how these proteins carry out their functions. To achieve these goals, proteomic approaches for the en masse identification of IDPs have been developed. 9,10 These almost invariably rely on an initial enrichment of IDPs by heat- induced or chemical denaturation of globular proteins, separa- tion of the of remaining proteins by 2D electrophoresis, and their subsequent identification by mass spectrometry (MS). A variant of these 2D-based approaches, described in our labora- tory, combines an initial native electrophoresis with a denatur- ing second one utilizing [8 M] urea. 11 MS identification of proteins along the diagonal line of the second gel provided evidence that this method not only separates IDPs from globular proteins, but also provides direct evidence for their structural status. Although this proteomic technique principally enables the identification of a large number of IDPs, we only used it to identify a handful of IDPs; thus, the concept has not been really substantiated. Further, in our initial attempt, the resolving power of the technique had not been really put to the test, because IDPs were identified from the proteomes of * To whom correspondence should be addressed at: Institute of Enzymol- ogy, Hungarian Academy of Sciences, 1113 Budapest, Karolina u ´t 29; tel, +361-279-3143; fax, +361-466-5465; e-mail, tompa@enzim.hu. † Institute of Enzymology, Hungarian Academy of Sciences. ‡ Research Institute for Solid State Physics and Optics of Hungarian Academy of Sciences. § Eo ¨tvo ¨s Lora ´nd University. | Proteomics Research Group, Biological Research Center, Hungarian Academy of Sciences. ⊥ University of California San Francisco. 10.1021/pr700720c CCC: $40.75  2008 American Chemical Society Journal of Proteome Research 2008, 7, 2291–2299 2291 Published on Web 05/17/2008