The Database of Protein Disorder (DisProt) links structure and function information for intrinsically disordered proteins (IDPs). Intrinsically disordered proteins do not form a fixed three-dimensional structure under physiological conditions, either in their entireties or in segments or regions. We define IDP as a protein that contains at least one experimentally determined disordered region. Although lacking fixed structure, IDPs and regions carry out important biological functions, being typically involved in regulation, signaling and control. Such functions can involve high-specificity low-affinity interactions, the multiple binding of one protein to many partners and the multiple binding of many proteins to one partner. These three features are all enabled and enhanced by protein intrinsic disorder. One of the major hindrances in the study of IDPs has been the lack of organized information. DisProt was developed to enable IDP research by collecting and organizing knowledge regarding the experimental characterization and the functional associations of IDPs. In addition to being a unique source of biological information, DisProt opens doors for a plethora of bioinformatics studies. DisProt is openly available at .

Alternative splicing of pre-mRNA generates two or more protein isoforms from a single gene, thereby contributing to protein diversity. Despite intensive efforts, an understanding of the protein structure-function implications of alternative splicing is still lacking. Intrinsic disorder, which is a lack of equilibrium 3D structure under physiological conditions, may provide this understanding. Intrinsic disorder is a common phenomenon, particularly in multicellular eukaryotes, and is responsible for important protein functions including regulation and signaling. We hypothesize that polypeptide segments affected by alternative splicing are most often intrinsically disordered such that alternative splicing enables functional and regulatory diversity while avoiding structural complications. We analyzed a set of 46 differentially spliced genes encoding experimentally characterized human proteins containing both structured and intrinsically disordered amino acid segments. We show that 81% of 75 alternatively spliced fragments in these proteins were associated with fully (57%) or partially (24%) disordered protein regions. Regions affected by alternative splicing were significantly biased toward encoding disordered residues, with a vanishingly small P value. A larger data set composed of 558 SwissProt proteins with known isoforms produced by 1,266 alternatively spliced fragments was characterized by applying the PONDR VSL1 disorder predictor. Results from prediction data are consistent with those obtained from experimental data, further supporting the proposed hypothesis. Associating alternative splicing with protein disorder enables the time-and tissue-specific modulation of protein function needed for cell differentiation and the evolution of multicellular organisms.evolution ͉ natively unfolded ͉ intrinsically unstructured ͉ protein structure T he splicing of pre-mRNA (1) was first described in 1977. Soon thereafter, Gilbert (2) coined the terms ''intron'' (intragenic region) and ''exon'' (expressed region) for the noncoding and coding regions, respectively. Alternative splicing occurs when different mRNAs are assembled from a single gene by joining exons in different ways. Alternative splicing is proposed to generate complexity in multicellular eukaryotes by increasing protein diversity, and thus proteome size, from a relatively small number of genes (3). Estimates indicate that between 35 and 60% of human genes yield protein isoforms by means of alternatively spliced (AS) mRNA (4). Furthermore, complexity in higher organisms is also brought about by signaling and regulatory networks that enable robustness (5). The importance of alternative splicing as a regulatory process (3, 6) has been highlighted by the high occurrence of such splicing in the pre-mRNAs of regulatory and signaling proteins (7).Alternative splicing can bolster organism complexity, not only by effectively increasing proteome size and regulatory and signaling network complexity, but also by doing so in a time-and tissue-specific manner, supporting ...

The identification of intrinsically disordered proteins (IDPs) among the targets that fail to form satisfactory crystal structures in the Protein Structure Initiative represent a key to reducing the costs and time for determining three-dimensional structures of proteins. To help in this endeavor, several Protein Structure Initiative Centers were asked to send samples of both crystallizable proteins and proteins that failed to crystallize. The abundance of intrinsic disorder in these proteins was evaluated via computational analysis using Predictors of Natural Disordered Regions (PONDR®) and the potential cleavage sites and corresponding fragments were determined. Then, the target proteins were analyzed for intrinsic disorder by their resistance to limited proteolysis. The rates of tryptic digestion of sample target proteins were compared to those of lysozyme/myoglobin, apo-myoglobin and α-casein as standards of ordered, partially disordered and completely disordered proteins, respectively. At the next stage, the protein samples were subjected to both far-UV and near-UV circular dichroism (CD) analysis. For most of the samples, a good agreement between CD data, predictions of disorder and the rates of limited tryptic digestion was established. Further experimentation is being performed on a smaller subset of these samples in order to obtain more detailed information on the ordered/disordered nature of the proteins.

immune system activation. Alterations in the regulation of CaN can lead to disorders such as Down syndrome-related mental retardation and cardiac hypertrophy. CaN is also the target for the immunosuppressant drugs FK506 and cyclosporin A. The regulation of CaN function is not well understood at the molecular level. CaN is inactive until bound by calmodulin (CaM). CaM binds at a site towards the N-terminus of a 95 residue regulatory domain in CaN. This regulatory domain is believed to be disordered. The binding of CaM to CaN causes an autoinhibitory domain located C-terminal to the regulatory domain to be ejected from CaN's active site. We hypothesize that the CaN regulatory domain undergoes a folding transition upon CaM binding, and that this folding provides the driving force for pulling the autoinhibitory domain from the active site. We have made a fragment of CaN that consists of the regulatory domain, autoinhibitory domain and a short C-terminal domain. We will present data from CD spectroscopy, fluorescence, NMR and analytical ultracentrifugation experiments that indicate this fragment is largely disordered in the absence of CaM, and gains structure when CaM binds.