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Association between foldability and aggregation propensity in small disulfide-rich proteins.

ABSTRACT: AIMS: Disulfide-rich domains (DRDs) are small proteins whose native structure is stabilized by the presence of covalent disulfide bonds. These domains are versatile and can perform a wide range of functions. Many of these domains readily unfold on disulfide bond reduction, suggesting that in the absence of covalent bonding they might display significant disorder. RESULTS: Here, we analyzed the degree of disorder in 97 domains representative of the different DRDs families and demonstrate that, in terms of sequence, many of them can be classified as intrinsically disordered proteins (IDPs) or contain predicted disordered regions. The analysis of the aggregation propensity of these domains indicates that, similar to IDPs, their sequences are more soluble and have less aggregating regions than those of other globular domains, suggesting that they might have evolved to avoid aggregation after protein synthesis and before they can attain its compact and covalently linked native structure. INNOVATION AND CONCLUSION: DRDs, which resemble IDPs in the reduced state and become globular when their disulfide bonds are formed, illustrate the link between protein folding and aggregation propensities and how these two properties cannot be easily dissociated, determining the main traits of the folding routes followed by these small proteins to attain their native oxidized states.

SUBMITTER: Fraga H

PROVIDER: S-EPMC4076991 | biostudies-literature | 2014 Jul

REPOSITORIES: biostudies-literature

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Association between foldability and aggregation propensity in small disulfide-rich proteins.

Fraga Hugo H Graña-Montes Ricardo R Illa Ricard R Covaleda Giovanni G Ventura Salvador S

Antioxidants & redox signaling 20140505 3

<h4>Aims</h4>Disulfide-rich domains (DRDs) are small proteins whose native structure is stabilized by the presence of covalent disulfide bonds. These domains are versatile and can perform a wide range of functions. Many of these domains readily unfold on disulfide bond reduction, suggesting that in the absence of covalent bonding they might display significant disorder.<h4>Results</h4>Here, we analyzed the degree of disorder in 97 domains representative of the different DRDs families and demonst ...[more]

PMID: 24635049

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Project description:BackgroundDisulfide-rich proteins or DRPs are versatile bioactive compounds that encompass a wide variety of pharmacological, therapeutic, and/or biotechnological applications. Still, the production of DRPs in sufficient quantities is a major bottleneck for their complete structural or functional characterization. Recombinant expression of such small proteins containing multiple disulfide bonds in the bacteria E. coli is considered difficult and general methods and protocols, particularly on a high throughput scale, are limited.ResultsHere we report a high throughput screening approach that allowed the systematic investigation of the solubilizing and folding influence of twelve cytoplasmic partners on 28 DRPs in the strains BL21 (DE3) pLysS, Origami B (DE3) pLysS and SHuffle® T7 Express lysY (1008 conditions). The screening identified the conditions leading to the successful soluble expression of the 28 DRPs selected for the study. Amongst 336 conditions tested per bacterial strain, soluble expression was detected in 196 conditions using the strain BL21 (DE3) pLysS, whereas only 44 and 50 conditions for soluble expression were identified for the strains Origami B (DE3) pLysS and SHuffle® T7 Express lysY respectively. To assess the redox states of the DRPs, the solubility screen was coupled with mass spectrometry (MS) to determine the exact masses of the produced DRPs or fusion proteins. To validate the results obtained at analytical scale, several examples of proteins expressed and purified to a larger scale are presented along with their MS and functional characterization.ConclusionsOur results show that the production of soluble and functional DRPs with cytoplasmic partners is possible in E. coli. In spite of its reducing cytoplasm, BL21 (DE3) pLysS is more efficient than the Origami B (DE3) pLysS and SHuffle® T7 Express lysY trxB(-)/gor(-) strains for the production of DRPs in fusion with solubilizing partners. However, our data suggest that oxidation of the proteins occurs ex vivo. Our protocols allow the production of a large diversity of DRPs using DsbC as a fusion partner, leading to pure active DRPs at milligram scale in many cases. These results open up new possibilities for the study and development of DRPs with therapeutic or biotechnological interest whose production was previously a limitation.

Dataset Information

Association between foldability and aggregation propensity in small disulfide-rich proteins.

Publications

Association between foldability and aggregation propensity in small disulfide-rich proteins.

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