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Sweeping away protein aggregation with entropic bristles: intrinsically disordered protein fusions enhance soluble expression.

ABSTRACT: Intrinsically disordered, highly charged protein sequences act as entropic bristles (EBs), which, when translationally fused to partner proteins, serve as effective solubilizers by creating both a large favorable surface area for water interactions and large excluded volumes around the partner. By extending away from the partner and sweeping out large molecules, EBs can allow the target protein to fold free from interference. Using both naturally occurring and artificial polypeptides, we demonstrate the successful implementation of intrinsically disordered fusions as protein solubilizers. The artificial fusions discussed herein have a low level of sequence complexity and a high net charge but are diversified by means of distinctive amino acid compositions and lengths. Using 6xHis fusions as controls, soluble protein expression enhancements from 65% (EB60A) to 100% (EB250) were observed for a 20-protein portfolio. Additionally, these EBs were able to more effectively solubilize targets compared to frequently used fusions such as maltose-binding protein, glutathione S-transferase, thioredoxin, and N utilization substance A. Finally, although these EBs possess very distinct physiochemical properties, they did not perturb the structure, conformational stability, or function of the green fluorescent protein or the glutathione S-transferase protein. This work thus illustrates the successful de novo design of intrinsically disordered fusions and presents a promising technology and complementary resource for researchers attempting to solubilize recalcitrant proteins.

SUBMITTER: Santner AA

PROVIDER: S-EPMC4141500 | biostudies-literature | 2012 Sep

REPOSITORIES: biostudies-literature

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Sweeping away protein aggregation with entropic bristles: intrinsically disordered protein fusions enhance soluble expression.

Santner Aaron A AA Croy Carrie H CH Vasanwala Farha H FH Uversky Vladimir N VN Van Ya-Yue J YY Dunker A Keith AK

Biochemistry 20120905 37

Intrinsically disordered, highly charged protein sequences act as entropic bristles (EBs), which, when translationally fused to partner proteins, serve as effective solubilizers by creating both a large favorable surface area for water interactions and large excluded volumes around the partner. By extending away from the partner and sweeping out large molecules, EBs can allow the target protein to fold free from interference. Using both naturally occurring and artificial polypeptides, we demonst ...[more]

PMID: 22924672

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Project description:Intrinsically disordered proteins (IDPs) refer to those proteins without fixed three-dimensional structures under physiological conditions. Although experiments suggest that the conformations of IDPs can vary from random coils, semi-compact globules, to compact globules with different contents of secondary structures, computational efforts to separate IDPs into different states are not yet successful. Recently, we developed a neural-network-based disorder prediction technique SPINE-D that was ranked as one of the top performing techniques for disorder prediction in the biannual meeting of critical assessment of structure prediction techniques (CASP 9, 2010). Here, we further analyze the results from SPINE-D prediction by defining a semi-disordered state that has about 50% predicted probability to be disordered or ordered. This semi-disordered state is partially collapsed with intermediate levels of predicted solvent accessibility and secondary structure content. The relative difference in compositions between semi-disordered and fully disordered regions is highly correlated with amyloid aggregation propensity (a correlation coefficient of 0.86 if excluding four charged residues and proline, 0.73 if not). In addition, we observed that some semi-disordered regions participate in induced folding, and others play key roles in protein aggregation. More specifically, a semi-disordered region is amyloidogenic in fully unstructured proteins (such as alpha-synuclein and Sup35) but prone to local unfolding that exposes the hydrophobic core to aggregation in structured globular proteins (such as SOD1 and lysozyme). A transition from full disorder to semi-disorder at about 30-40 Qs is observed in the poly-Q (poly-glutamine) tract of huntingtin. The accuracy of using semi-disorder to predict binding-induced folding and aggregation is compared with several methods trained for the purpose. These results indicate the usefulness of three-state classification (order, semi-disorder, and full-disorder) in distinguishing nonfolding from induced-folding and aggregation-resistant from aggregation-prone IDPs and in locating weakly stable, locally unfolding, and potentially aggregation regions in structured proteins. A comparison with five representative disorder-prediction methods showed that SPINE-D is the only method with a clear separation of semi-disorder from ordered and fully disordered states.

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Sweeping away protein aggregation with entropic bristles: intrinsically disordered protein fusions enhance soluble expression.

Publications

Sweeping away protein aggregation with entropic bristles: intrinsically disordered protein fusions enhance soluble expression.

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