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

Graether, Steffen P.

doi:10.3389/fmolb.2018.00118

Cited by 18 publications

(12 citation statements)

References 74 publications

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“…A number of missing or overlapping residues was observed in the SUMO-G0S2 NMR spectrum around the intrinsically disordered region (Fig 5) [20], which is consistent with previous prediction [20,41]. This problem can be overcome by conducting CON series of experiments [42] in addition to the collected spectrum as shown in S5 Fig. The combination of results from current measurements and CON series of experiments can lead to more reliable residue assignments by taking advantage of the slow relaxation and the large chemical shift dispersion for analyzing these experimental results.…”

Section: Plos Onesupporting

confidence: 88%

Biophysical characterization and a roadmap towards the NMR solution structure of G0S2, a key enzyme in non-alcoholic fatty liver disease

et al. 2021

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In the United States non-alcoholic fatty liver disease (NAFLD) is the most common form of chronic liver disease, affecting an estimated 80 to 100 million people. It occurs in every age group, but predominantly in people with risk factors such as obesity and type 2 diabetes. NAFLD is marked by fat accumulation in the liver leading to liver inflammation, which may lead to scarring and irreversible damage progressing to cirrhosis and liver failure. In animal models, genetic ablation of the protein G0S2 leads to alleviation of liver damage and insulin resistance in high fat diets. The research presented in this paper aims to aid in rational based drug design for the treatment of NAFLD by providing a pathway for a solution state NMR structure of G0S2. Here we describe the expression of G0S2 in an E. coli system from two different constructs, both of which are confirmed to be functionally active based on the ability to inhibit the activity of Adipose Triglyceride Lipase. In one of the constructs, preliminary NMR spectroscopy measurements show dominant alpha-helical characteristics as well as resonance assignments on the N-terminus of G0S2, allowing for further NMR work with this protein. Additionally, the characterization of G0S2 oligomers are outlined for both constructs, suggesting that G0S2 may defensively exist in a multimeric state to protect and potentially stabilize the small 104 amino acid protein within the cell. This information presented on the structure of G0S2 will further guide future development in the therapy for NAFLD.

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Section: Plos Onesupporting

confidence: 88%

Biophysical characterization and a roadmap towards the NMR solution structure of G0S2, a key enzyme in non-alcoholic fatty liver disease

et al. 2021

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“…For cloned genes containing rare codons, E. coli strains such as Rosetta (DE3) may be a more appropriate choice. In some cases, the expressed IDPs are insoluble and may end up in inclusion bodies, and other vectors with different tags may be needed (Graether, 2019).…”

Section: Notesmentioning

confidence: 99%

“…We describe here a protocol to produce an isotopically labeled, fully disordered protein for detailed NMR experiments using an E. coli recombinant expression system (Graether, 2019). The protocol uses a plant stress protein that is studied in our laboratory as an example, the late embryogenesis abundant 3-2 protein from Arabidopsis thaliana (AtLEA 3-2, AT3G53770.1).…”

Section: Introductionmentioning

confidence: 99%

Expression and Purification of an Intrinsically Disordered Protein

Singh

Graether

2020

Methods in Molecular Biology

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LEA3 proteins are intrinsically disordered proteins expressed during seed development and in response to abiotic stress. Limited studies have shown they can confer stress tolerance in plants, yet their mechanism remains elusive. This thesis aims to further our understanding of the LEA3 group. First, a comprehensive bioinformatics analysis revealed two previously undiscovered C-terminal motifs containing conserved acidic and hydrophobic residues and four N-terminal motifs. Five general architectures were proposed for LEA3 and the physiochemical properties of the different architectures showed clustering in a relatively narrow range compared to the previously studied dehydrins. The evolutionary analysis revealed that the proteins grouped into clades based on their architecture, and that there appears to be at least two distinct groups of LEA3 proteins based on their architectures and physiochemical properties. The presence of LEA3 proteins in non-vascular plants but their absence in algae suggests that LEA3 may have arose in the evolution of land plants. A protocol to express and purify AtLEA3-2 with 15 N and 13 C isotopes in E. coli is described, although the protocol can be adapted for any LEA3 with or without isotopic labeling. The AtLEA 3-2 gene was cloned into the pET-SUMO vector, which allowed for the SUMO-AtLEA 3-2 fusion protein to be purified using Ni-affinity chromatography and, through the use of Ulp1, a

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“…SAXS is commonly used for the characterization of the low-resolution structure of macromolecules in solution [70,71]. Importantly, SAXS is especially useful for the analysis of the IDPs with elongated and exible chains, where other methods fail [72]. Therefore, we performed SAXS studies to get additional information regarding the structure and conformational dynamics of GCEC in solution.…”

Section: Saxs Analysismentioning

confidence: 99%

The Intrinsically Disordered Region of GCE Protein Adopts a More Fixed Structure by Interacting With the LBD of the Nuclear Receptor FTZ-F1

Kolonko

Bystranowska

Taube

et al. 2020

Preprint

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The Drosophila melanogaster Germ cell-expressed protein (GCE) is a paralog of the juvenile hormone (JH) receptor - Methoprene tolerant protein (MET). Both proteins mediate JH function, preventing precocious differentiation during D. melanogaster development. Despite that GCE and MET are often referred to as equivalent JH receptors, their functions are not fully redundant and show tissue specificity. Both proteins belong to the family of bHLH-PAS transcription factors. The similarity of their primary structure is limited to defined bHLH and PAS domains, while their long C-terminal fragments (GCEC, METC) show significant differences and are expected to determine differences in GCE and MET protein activities. In this paper we present the structural characterization of GCEC as a coil-like intrinsically disordered protein (IDP) with highly elongated and asymmetric conformation. In comparison to previously characterized METC, GCEC is less compacted, contains more molecular recognition elements (MoREs) and exhibits a higher propensity for induced folding. The NMR shifts perturbation experiment and pull-down assay clearly demonstrated that the GCEC fragment is sufficient to form an interaction interface with the ligand binding domain (LBD) of the nuclear receptor Fushi Tarazu factor-1 (FTZ-F1). Significantly, these interactions can force GCEC to adopt more fixed structure that can modulate the activity, structure and functions of the full-length receptor. The discussed relation of protein functionality with the structural data of inherently disordered GCEC fragment is a novel look at this protein and contributes to a better understanding of the molecular basis of the functions of the C-terminal fragments of the bHLH-PAS family.

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

Cited by 18 publications

References 74 publications

Biophysical characterization and a roadmap towards the NMR solution structure of G0S2, a key enzyme in non-alcoholic fatty liver disease

Biophysical characterization and a roadmap towards the NMR solution structure of G0S2, a key enzyme in non-alcoholic fatty liver disease

Expression and Purification of an Intrinsically Disordered Protein

The Intrinsically Disordered Region of GCE Protein Adopts a More Fixed Structure by Interacting With the LBD of the Nuclear Receptor FTZ-F1

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