High-throughput characterization of intrinsic disorder in proteins from the Protein Structure Initiative

Abstract

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.

Characterization of the Grp94/OS-9 Chaperone–Lectin Complex

“…Control digests were carried out on Grp94-M (residues 337–629), which is well folded, and α-casein, which is intrinsically disordered. 42 As seen in Figure 7E, OS-9 ΔMRH exhibits little resistance to limited tryptic digest. Substantial cutting was observed at the intermediate trypsin concentration and complete digestion of the full length polypeptide was seen at the highest trypsin concentration.…”

A Group 6 Late Embryogenesis Abundant Protein from Common Bean Is a Disordered Protein with Extended Helical Structure and Oligomer-forming Properties

“…The three isodichroic points exhibited by this analysis suggested changes in the local environment of more than one amino acid residue in this protein, possibly those corresponding to Phe and to one or more of the Tyr residues. The isodichroic points at 271 and 282 nm are in a region corresponding to Tyr signals (50), suggesting that some of the Tyr residues may be in equilibrium between three conformational stages or, alternatively, two Tyr in equilibrium with two conformations. These profiles are consistent with temperature-induced changes in the structure of this protein as suggested by far-UV CD spectroscopy.…”

Paradoxes and wonders of intrinsic disorder: Stability of instability

“…59 This structural instability is supported by the well-known fact of high sensitivity of IDPs/IDPRs to proteolytic degradation. [68][69][70][71][72][73][74][75][76][77] High resilience of intrinsic disorder Although lacking stable structure, possessing noncooperative unfolding behavior, and showing high sensitivity to proteolysis, one of the most intriguing biophysical properties ascribed to highly disordered proteins is their extraordinary resilience, where an IDP can sustain exposure to the extremely harsh environmental conditions, being able either to keep its functionality under these extreme conditions or to rapidly regain it after returning to normal conditions. 48,49 An illustrative example of such behavior is given by a "funny protein" prothymosin a, 48 which triggered my interest to the intrinsically disordered proteins by its unusual ability to be unharmed by the prolonged exposure to harsh conditions (activity of the protein was not affected by boiling for a few days).…”