Near-native, site-specific and purification-free protein labeling for quantitative protein interaction analysis by MicroScale Thermophoresis

Bartoschik, Tanja; Galinec, Stefanie; Kleusch, Christian; Walkiewicz, Katarzyna; Breitsprecher, Dennis; Weigert, Sebastian; Muller, Yves A.; You, Changjiang; Piehler, Jacob; Vercruysse, Thomas; Daelemans, Dirk; Tschammer, Nuška

doi:10.1038/s41598-018-23154-3

Cited by 65 publications

(46 citation statements)

References 41 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Immediately after IMAC elution, IR-ECD was directly applied to a Superdex 200 Increase 300/10 GL column (GE Healthcare) at 0.5 ml/min in HBS at room temperature and the two peak fractions were pooled for ligand binding assays. IR-ECD was diluted to a final concentration of 100 nM in HBS-T (HBS, pH 7.5, 0.05% Tween-20) and labelled with 25 nM tris-nitriloacetic acid conjugated to NT647 (RED-tris-NTA) (Lata et al, 2005;Bartoschik et al, 2018), which was a kind gift of Jacob Piehler (University Osnabrück, Germany). A dye-to-protein molar ratio of 1:4 was chosen to circumvent interference of free dye.…”

Section: Microscale Thermophoresis To Determine Insulin Binding To Irmentioning

confidence: 99%

Cryo-EM structure of the complete and ligand-saturated insulin receptor ectodomain

Gutmann

Schäfer

Poojari³

et al. 2019

Preprint

View full text Add to dashboard Cite

Glucose homeostasis and growth essentially depend on the peptide hormone insulin engaging its receptor. Despite biochemical and structural advances, a fundamental contradiction has persisted in the current understanding of insulin ligand-receptor interactions. While biochemistry predicts two distinct insulin binding sites, 1 and 2, recent structural analyses have only resolved site 1.Using a combined approach of cryo-EM and atomistic molecular dynamics simulation, we determined the structure of the entire dimeric insulin receptor ectodomain saturated with four insulin molecules. Complementing the previously described insulin-site 1 interaction, we present the first view of insulin bound to the discrete insulin receptor site 2. Insulin binding stabilizes the receptor ectodomain in a T-shaped conformation wherein the membrane-proximal domains converge and contact each other. These findings expand the current models of insulin binding to its receptor and of its regulation. In summary, we provide the structural basis enabling a comprehensive description of ligand-receptor interactions that ultimately will inform new approaches to structure-based drug design. In briefA cryo-EM structure of the complete insulin receptor ectodomain saturated with four insulin ligands is reported. The structural model of the insulin-insulin receptor complex adopts a T-shaped conformation, reveals two additional insulin-binding sites potentially involved in the initial interaction of insulin with its receptor, and resolves the membrane proximal region.

show abstract

Section: Microscale Thermophoresis To Determine Insulin Binding To Irmentioning

confidence: 99%

Cryo-EM structure of the complete and ligand-saturated insulin receptor ectodomain

Gutmann

Schäfer

Poojari³

et al. 2019

Preprint

View full text Add to dashboard Cite

show abstract

“…MST experiments were performed at 24°C on a NanoTemper® Monolith NT.115 instrument with red filters, using 40% LED power and 60% MST power. All the experiments were carried out using 6His-tagged HMGB1, non-covalently labelled with the NT647 fluorescence dye (Bartoschik et al, 2018). MST buffer contained 20 mM phosphate buffer pH 7.3, 150 mM NaCl, 0.05% Tween, 1 mM DTT.…”

Section: Mst Experimentsmentioning

confidence: 99%

Diflunisal targets the HMGB1/CXCL12 heterocomplex and blocks immune cell recruitment

Leo

Quilici²,

Tirone

et al. 2019

Preprint

View full text Add to dashboard Cite

Extracellular HMGB1 triggers inflammation following infection or injury, and supports tumorigenesis in inflammation-related malignancies. HMGB1 has several redox states: reduced HMGB1 recruits inflammatory cells to injured tissues forming a heterocomplex with CXCL12 and signaling via its receptor CXCR4; disulfide-containing HMGB1 binds to TLR4 and promotes inflammatory responses. Here we show that Diflunisal, an aspirin-like nonsteroidal anti-inflammatory drug (NSAID) that has been in clinical use for decades, specifically inhibits in vitro and in vivo the chemotactic activity of HMGB1 at nanomolar concentrations, at least in part by binding directly to both HMGB1 and CXCL12 and disrupting their heterocomplex. Importantly, Diflunisal does not inhibit TLR4-dependent responses. Our findings clarify the mode of action of Diflunisal, and open the way to the rational design of functionally specific anti-inflammatory drugs.Diflunisal targets HMGB1-CXCL12 complex

show abstract

“…Microscale thermophoresis is a new technique to study the interactions of proteins and small molecules with several advantages over other technologies (Bartoschik et al., 2018; Wienken, Baaske, Rothbauer, Braun, & Duhr, 2010). One of significant advantages is its capability to measure dissociation constants ( K d s) in solution with low consumption of sample.…”

Section: Methodsmentioning

confidence: 99%

Polysaccharide selection and mechanism for prevention of protein–polyphenol haze formation in beverages

Wang

Zhao

Liao

et al. 2020

Journal of Food Science

View full text Add to dashboard Cite

Polysaccharides have been considered as a group of promising candidate for preventing the protein–polyphenol haze formation in beverages. In order to select effective polysaccharides to prevent the haze formation, four protein–polyphenol haze model systems were successfully established using two proteins (i.e., gelatin and bovine serum albumin) and two polyphenols (i.e., procyanidin [PC] and epigallocatechin gallate [EGCG]). Among seven common polysaccharides, 0.5 mg/mL pectin, 0.05 mg/mL xanthan gum, and 0.01 mg/mL guar gum demonstrated the maximum potential for preventing the formation of four protein–polyphenol hazes. Ultraviolet‐visible spectrophotometry confirmed that polysaccharides affected protein–polyphenol interactions. Fluorescence spectrophotometry combined with microscale thermophoresis data indicated the relative affinities of polyphenol to protein and polysaccharide determined the mechanism of polysaccharide for preventing the haze formation. In bovine serum albumin (BSA)/gelatin‐EGCG system, polysaccharides (pectin, xanthan gum and guar gum) competed with BSA/gelatin to bind EGCG for prevention the formation of BSA/gelatin‐EGCG haze. However, in BSA/gelatin‐PC system, polysaccharides (pectin, xanthan gum, and guar gum) formed a ternary complex (protein–tannin–polysaccharide) for increasing the solubility of protein‐polyphenol aggregation. From apple juice results, the reduction rates of guar gum in two apple juice systems (gelatin‐PC, BSA‐PC) were 21% and 56% within 8 weeks, indicating guar gum might be the most effective polysaccharide in preventing the haze formation. Practical Application This experiment data could be used for development of polysaccharide products for prevention of protein–polyphenol haze formation in beverages.

show abstract

Near-native, site-specific and purification-free protein labeling for quantitative protein interaction analysis by MicroScale Thermophoresis

Cited by 65 publications

References 41 publications

Cryo-EM structure of the complete and ligand-saturated insulin receptor ectodomain

Cryo-EM structure of the complete and ligand-saturated insulin receptor ectodomain

Diflunisal targets the HMGB1/CXCL12 heterocomplex and blocks immune cell recruitment

Polysaccharide selection and mechanism for prevention of protein–polyphenol haze formation in beverages

Contact Info

Product

Resources

About