Production of Intrinsically Disordered Proteins for Biophysical Studies: Tips and Tricks

Pedersen, Christian Parsbæk; Seiffert, Pernille; Brakti, Inna; Bugge, Katrine

doi:10.1007/978-1-0716-0524-0_9

Cited by 15 publications

(17 citation statements)

References 46 publications

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“…Spd1 and Spd1-4G (L33G, V36G, F55G and Y58G) were expressed and purified from 15 N-labeled ( 15 (NH 4 ) 2 SO 4 ) of 15 N and 13 C labeled ( 13 C 6 -glucose) M9 medium as previously described [42]. The Spd1-CTD (Spd1 59-124 ) was expressed from a pET24b vector coupled to a His 6 -SUMO tag and purified following standard protocols [43]. The sample was purified further by the means of reverse phase chromatography (RPC).…”

Section: Expression and Purification Of Spd1 And Variantsmentioning

confidence: 99%

A context-dependent and disordered ubiquitin-binding motif

Dreier

Prestel

Martins

et al. 2022

Cell. Mol. Life Sci.

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Ubiquitin is a small, globular protein that is conjugated to other proteins as a posttranslational event. A palette of small, folded domains recognizes and binds ubiquitin to translate and effectuate this posttranslational signal. Recent computational studies have suggested that protein regions can recognize ubiquitin via a process of folding upon binding. Using peptide binding arrays, bioinformatics, and NMR spectroscopy, we have uncovered a disordered ubiquitin-binding motif that likely remains disordered when bound and thus expands the palette of ubiquitin-binding proteins. We term this motif Disordered Ubiquitin-Binding Motif (DisUBM) and find it to be present in many proteins with known or predicted functions in degradation and transcription. We decompose the determinants of the motif showing it to rely on features of aromatic and negatively charged residues, and less so on distinct sequence positions in line with its disordered nature. We show that the affinity of the motif is low and moldable by the surrounding disordered chain, allowing for an enhanced interaction surface with ubiquitin, whereby the affinity increases ~ tenfold. Further affinity optimization using peptide arrays pushed the affinity into the low micromolar range, but compromised context dependence. Finally, we find that DisUBMs can emerge from unbiased screening of randomized peptide libraries, featuring in de novo cyclic peptides selected to bind ubiquitin chains. We suggest that naturally occurring DisUBMs can recognize ubiquitin as a posttranslational signal to act as affinity enhancers in IDPs that bind to folded and ubiquitylated binding partners.

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Section: Expression and Purification Of Spd1 And Variantsmentioning

confidence: 99%

A context-dependent and disordered ubiquitin-binding motif

Dreier

Prestel

Martins

et al. 2022

Cell. Mol. Life Sci.

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“…Protein purification. All four variants of S. pombe Dss1 were designed to encode an Nterminal His6-SUMO tag to be cleaved with ubiquitin-like protein protease 1 (ULP1) following initial purification with a nickel column [29]. All four variants were purified with isotope labelling as described in previous work [30], resulting in lyophilized pure protein, ready for resuspension in the buffer of choice.…”

Section: Methodsmentioning

confidence: 99%

Deciphering the alphabet of disorder — Glu and Asp act differently on local but not global properties

Roesgaard

Lundsgaard

Newcombe

et al. 2022

Preprint

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Compared to folded proteins, the sequences of intrinsically disordered proteins (IDPs) are enriched in polar and charged amino acids. Glutamate is one of the most enriched amino acids in IDPs, while the chemically similar amino acid aspartate is less enriched. So far, the underlying functional differences of glutamates and aspartates in IDPs remain poorly understood. In this study, we examine the differential effects of aspartate and glutamates in IDPs by comparing the function and conformational ensemble of glutamate and aspartate variants of the disordered protein Dss1, using a range of assays, including interaction studies, nuclear magnetic resonance spectroscopy, small angle X-ray scattering and molecular dynamics simulation. First, we analyze the sequences of the rapidly growing data base of experimentally verified IDPs (DisProt) and show that the glutamate enrichment is not caused by a taxonomy bias in IDPs. From analyses of local and global structural properties as well as cell growth and protein-protein interactions using model acidic IDP from yeast and three Glu/Asp variants, we find that while Glu/Asp support similar function and global dimensions, the variants differ in their binding affinities and population of local transient structural elements. We speculate that these local structural differences may play roles in functional diversity where glutamates can support increased helicity important for folding and binding, while aspartates support extended structures and form helical caps, as well as playing more relevant roles in e.g., transactivation domains and ion-binding.

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“…All constructs (proteins sequences Table S1), except those encoding the C-terminal region of the human sodium proton exchange 1 (NHE1 680-815 ), human ProTα, and fulllength human aSN (aSN 1-140 ), were designed to encode an N-terminal His 6 -SUMO tag, to be cleaved with ubiquitin-like protein protease 1 (ULP1) following initial purification with a nickel column [26]. E. coli cells were transformed with one of the following plasmids using heat shock transformation: His 6 -SUMO-aSN 96-140 (BL21 DE3), His 6 -SUMO-ANAC046 172-338 (BL21-codon plus DE3; the protein represents the C-terminal region of the Arabidopsis thaliana ANA046 protein (ANAC: Arabidopsis no apical meristem [NAM], Arabidopsis transcription activation factor [ATAF], and cup-shaped cotyledon [CUC])), and His 6 -SUMO-DSS1 (BL21 DE3; Schizosaccharomyces pombe deleted in split hand/split foot 1).…”

Section: General Protein and Peptide Productionmentioning

confidence: 99%

“…E. coli cells were transformed with one of the following plasmids using heat shock transformation: His 6 -SUMO-aSN 96-140 (BL21 DE3), His 6 -SUMO-ANAC046 172-338 (BL21-codon plus DE3; the protein represents the C-terminal region of the Arabidopsis thaliana ANA046 protein (ANAC: Arabidopsis no apical meristem [NAM], Arabidopsis transcription activation factor [ATAF], and cup-shaped cotyledon [CUC])), and His 6 -SUMO-DSS1 (BL21 DE3; Schizosaccharomyces pombe deleted in split hand/split foot 1). In each case, pre-cultures (10 mL; LB medium kanamycin 50 µg/mL) were used to inoculate M9-minimal medium (1 L) [26,27], containing 15 NH 4 Cl (1 g/L), 13 C 6 -glucose (4 g/L), and kanamycin (50 µg/mL). For specific growth times and induction details, see Table S2.…”

Section: General Protein and Peptide Productionmentioning

confidence: 99%

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Insight into Calcium-Binding Motifs of Intrinsically Disordered Proteins

et al. 2021

Self Cite

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Motifs within proteins help us categorize their functions. Intrinsically disordered proteins (IDPs) are rich in short linear motifs, conferring them many different roles. IDPs are also frequently highly charged and, therefore, likely to interact with ions. Canonical calcium-binding motifs, such as the EF-hand, often rely on the formation of stabilizing flanking helices, which are a key characteristic of folded proteins, but are absent in IDPs. In this study, we probe the existence of a calcium-binding motif relevant to IDPs. Upon screening several carefully selected IDPs using NMR spectroscopy supplemented with affinity quantification by colorimetric assays, we found calcium-binding motifs in IDPs which could be categorized into at least two groups—an Excalibur-like motif, sequentially similar to the EF-hand loop, and a condensed-charge motif carrying repetitive negative charges. The motifs show an affinity for calcium typically in the ~100 μM range relevant to regulatory functions and, while calcium binding to the condensed-charge motif had little effect on the overall compaction of the IDP chain, calcium binding to Excalibur-like motifs resulted in changes in compaction. Thus, calcium binding to IDPs may serve various structural and functional roles that have previously been underreported.

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Production of Intrinsically Disordered Proteins for Biophysical Studies: Tips and Tricks

Cited by 15 publications

References 46 publications

A context-dependent and disordered ubiquitin-binding motif

A context-dependent and disordered ubiquitin-binding motif

Deciphering the alphabet of disorder — Glu and Asp act differently on local but not global properties

Insight into Calcium-Binding Motifs of Intrinsically Disordered Proteins

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