Recombinant Intrinsically Disordered Proteins for NMR: Tips and Tricks

Calçada, Eduardo O.; Korsak, Magdalena; Kozyreva, Tatiana

doi:10.1007/978-3-319-20164-1_6

Cited by 7 publications

(5 citation statements)

References 65 publications

(61 reference statements)

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“…Firstly, when separating the N-terminus (Nt) of SS2 by GPC, it eluted in fractions corresponding to relatively large oligomers containing at least 6-8 subunits (Figure 6 ) When expressed recombinantly, intrinsically disordered proteins (IDP) are prone to aggregation (Lebendiker and Danieli, 2014 ). When separated on SDS-PAGE, IDP/IDR's typically migrate as though they exhibit a higher molecular mass (Calçada et al, 2015 ). This abnormal SDS-PAGE migration pattern was seen for the wild type SS2 proteins, which has a predicted molecular weight of 85 kDa, but migrated close to the 100 kDa marker (Figures 4 – 6 ), and has previously been observed for the SSIIa isoform in wheat endosperm (Li et al, 1999 ).…”

Section: Discussionmentioning

confidence: 99%

Bioinformatic and in vitro Analyses of Arabidopsis Starch Synthase 2 Reveal Post-translational Regulatory Mechanisms

2018

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Starch synthase 2 (SS2) is an important enzyme in leaf starch synthesis, elongating intermediate-length glucan chains. Loss of SS2 results in a distorted starch granule phenotype and altered physiochemical properties, highlighting its importance in starch biosynthesis, however, the post-translational regulation of SS2 is poorly understood. In this study, a combination of bioinformatic and in vitro analysis of recombinant SS2 was used to identify and characterize SS2 post-translational regulatory mechanisms. The SS2 N-terminal region, comprising the first 185 amino acids of the mature protein sequence, was shown to be highly variable between species, and was predicted to be intrinsically disordered. Intrinsic disorder in proteins is often correlated with protein phosphorylation and protein-protein interactions. Recombinant Arabidopsis thaliana SS2 formed homodimers that required the N-terminal region, but N-terminal peptides could not form stable homodimers alone. Recombinant SS2 was shown to be phosphorylated by chloroplast protein kinases and recombinant casein kinase II at two N-terminal serine residues (S63, S65), but mutation of these phosphorylation sites (Ser>Ala) revealed that they are not required for homo-dimerization. Heteromeric enzyme complex (HEC) formation between SS2 and SBE2.2 was shown to be ATP-dependent. However, SS2 homo-dimerization and protein phosphorylation are not required for its interaction with SBE2.2, as truncation of the SS2 N-terminus did not disrupt ATP-dependent HEC assembly. SS2 phosphorylation had no affect on its catalytic activity. Intriguingly, the removal of the N-terminal region of SS2 resulted in a 47-fold increase in its activity. As N-terminal truncation disrupted dimerization, this suggests that SS2 is more active when monomeric, and that transitions between oligomeric state may be a mechanism for SS2 regulation.

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Section: Discussionmentioning

confidence: 99%

Bioinformatic and in vitro Analyses of Arabidopsis Starch Synthase 2 Reveal Post-translational Regulatory Mechanisms

2018

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“…The source DNA may be genomic, and need to be PCR amplified and manipulated using routine molecular biological approaches to incorporate it into a plasmid. One method, while not new but becoming increasingly affordable, is the “clone-by-phone” approach (Calçada et al, 2015), where the protein sequence is submitted to a commercial service, and for a fee a plasmid is sent in return. A major advantage of this approach is that the sequence can be optimized for recombinant host expression, which is not necessarily the same as the DNA source species.…”

Section: Gene Design and Recombinant Expressionmentioning

confidence: 99%

“…A researcher may wish to screen several different plasmids with different tags encoded in the plasmid to facilitate expression and purification. In this case, it is best to consider a high throughput system that uses ligase independent cloning methods (e.g., Gateway or TOPO) to simplify and accelerate the cloning process (Calçada et al, 2015).…”

Section: Gene Design and Recombinant Expressionmentioning

confidence: 99%

Troubleshooting Guide to Expressing Intrinsically Disordered Proteins for Use in NMR Experiments

Graether

2019

Front. Mol. Biosci.

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Intrinsically disordered proteins (IDPs) represent a structural class of proteins that do not have a well-defined, 3D fold in solution, and often have little secondary structure. To characterize their function and molecular mechanism, it is helpful to examine their structure using nuclear magnetic resonance (NMR), which can report on properties, such as residual structure (at both the secondary and tertiary levels), ligand binding affinity, and the effect of ligand binding on IDP structure, all on a per residue basis. This brief review reports on the common problems and decisions that are involved when preparing a disordered protein for NMR studies. The paper covers gene design, expression host choice, protein purification, and the initial NMR experiments that are performed. While many of these steps are essentially identical to those for ordered proteins, a few key differences are highlighted, including the extreme sensitivity of IDPs to proteolytic cleavage, the ability to use denaturing conditions without having to refold the protein, the optimal chromatographic system choice, and the challenges of quantifying an IDP. After successful purification, characterization by NMR can be done using the standard 15N-heteronuclear single quantum coherence (15N-HSQC) experiment, or the newer CON series of experiments that are superior for disordered proteins.

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“…They are frequently associated with disease. For these reasons, there is growing interest in IDRs as potential targets for drug design (Calcada, Korsak & Kozyreva, 2015; Cheng et al, 2006). The prediction of 14 flexible disordered regions in GiK suggests that this protein may be important in various Giardia functions.…”

Section: Discussionmentioning

confidence: 99%

Identification of a novel potassium channel (GiK) as a potential drug target in Giardia lamblia: Computational descriptions of binding sites

Palomo‐Ligas

Gutiérrez-Gutiérrez

Ochoa-Maganda

et al. 2019

PeerJ

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Background The protozoan Giardia lamblia is the causal agent of giardiasis, one of the main diarrheal infections worldwide. Drug resistance to common antigiardial agents and incidence of treatment failures have increased in recent years. Therefore, the search for new molecular targets for drugs against Giardia infection is essential. In protozoa, ionic channels have roles in their life cycle, growth, and stress response. Thus, they are promising targets for drug design. The strategy of ligand-protein docking has demonstrated a great potential in the discovery of new targets and structure-based drug design studies. Methods In this work, we identify and characterize a new potassium channel, GiK, in the genome of Giardia lamblia. Characterization was performed in silico. Because its crystallographic structure remains unresolved, homology modeling was used to construct the three-dimensional model for the pore domain of GiK. The docking virtual screening approach was employed to determine whether GiK is a good target for potassium channel blockers. Results The GiK sequence showed 24–50% identity and 50–90% positivity with 21 different types of potassium channels. The quality assessment and validation parameters indicated the reliability of the modeled structure of GiK. We identified 110 potassium channel blockers exhibiting high affinity toward GiK. A total of 39 of these drugs bind in three specific regions. Discussion The GiK pore signature sequence is related to the small conductance calcium-activated potassium channels (SKCa). The predicted binding of 110 potassium blockers to GiK makes this protein an attractive target for biological testing to evaluate its role in the life cycle of Giardia lamblia and potential candidate for the design of novel antigiardial drugs.

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Recombinant Intrinsically Disordered Proteins for NMR: Tips and Tricks

Cited by 7 publications

References 65 publications

Bioinformatic and in vitro Analyses of Arabidopsis Starch Synthase 2 Reveal Post-translational Regulatory Mechanisms

Bioinformatic and in vitro Analyses of Arabidopsis Starch Synthase 2 Reveal Post-translational Regulatory Mechanisms

Troubleshooting Guide to Expressing Intrinsically Disordered Proteins for Use in NMR Experiments

Identification of a novel potassium channel (GiK) as a potential drug target in Giardia lamblia: Computational descriptions of binding sites

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