Bacterial Expression and In Vitro Refolding of Limited Fragments of the Notch Receptor and Its Ligands

Whiteman, Pat; Redfield, Christina; Handford, Penny A.

doi:10.1007/978-1-4939-1139-4_15

Cited by 5 publications

(5 citation statements)

References 14 publications

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“…Protein expression, isotopic labeling, refolding, and purification protocols have been described previously ( Muranyi et al., 2004 , Weisshuhn et al., 2015a , Weisshuhn et al., 2015b , Whiteman et al., 2014 ). Protein fragments were expressed in Escherichia coli BL21 cells transformed with a pQE30 (Qiagen)-based protein expression construct and a pREP4 plasmid for control of expression via the Lac Repressor.…”

Section: Methodsmentioning

confidence: 99%

Non-Linear and Flexible Regions of the Human Notch1 Extracellular Domain Revealed by High-Resolution Structural Studies

et al. 2016

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SummaryThe Notch receptor is a key component of a core metazoan signaling pathway activated by Delta/Serrate/Lag-2 ligands expressed on an adjacent cell. This results in a short-range signal with profound effects on cell-fate determination, cell proliferation, and cell death. Key to understanding receptor function is structural knowledge of the large extracellular portion of Notch which contains multiple repeats of epidermal growth factor (EGF)-like domains. Here we investigate the EGF4-13 region of human Notch1 (hN1) using a multidisciplinary approach. Ca2+-binding measurements, X-ray crystallography, {1H}-15N heteronuclear nuclear Overhauser effects, and residual dipolar couplings support a non-linear organization for the EGF4-13 region with a rigid, bent conformation for EGF4-7 and a single flexible linkage between EGF9 and EGF10. These data allow us to construct an informed model for EGF10-13 which, in conjunction with comparative binding studies, demonstrates that EGF10 has an important role in determining Notch receptor sensitivity to Dll-4.

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Section: Methodsmentioning

confidence: 99%

Non-Linear and Flexible Regions of the Human Notch1 Extracellular Domain Revealed by High-Resolution Structural Studies

et al. 2016

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“…Protein expression, purification and refolding Protein expression and purification was carried out as described previously for similar constructs (Weisshuhn et al 2014;Knott et al 1996;Whiteman et al 2014). Constructs Schematic pictures of the EGF8-11, EGF9-11 and EGF11-13 constructs, discussed in this study, are shown.…”

Section: Methods and Experimentsmentioning

confidence: 99%

1H, 13C and 15N assignments of EGF domains 8–11 of human Notch-1

2015

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The Notch receptor is part of a core cell-cell signaling system crucial for development and tissue homeostasis in Metazoa. Structural information is available for the negative regulatory region, the ligand-binding region and the intracellular domain of Notch, but data for the remaining portions of the extracellular region which determine its overall shape at the cell surface are still lacking. This region consists of 36 EGF-like domains arranged as multiple tandem repeats. Most EGF-like domains near the ligand-binding domains EGF11 and 12 are of the calcium-binding type, with well-described, rigid and near-linear interdomain interfaces. However, EGF10 is a conserved, non-calcium-binding domain which may confer flexibility or a non-linear organization to the receptor. To probe this, we have expressed and purified a four-domain construct, EGF8-11, from human Notch-1, and report here the (1)H, (13)C and (15)N resonance assignments. Differences in EGF11 chemical shifts between this construct and a previously assigned construct, EGF11-13, confirm the presence of hydrophobic interdomain contacts between the hairpin turn of the major β-sheet in EGF11 and the conserved aromatic residue within the C-terminal region of EGF10. This suggests that the EGF10-11 interface is rigid.

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“…Protein expression, purification and refolding Protein expression and purification was carried out as described previously for similar constructs (Weisshuhn et al, 2014, Knott et al 1996, Whiteman, Redfield, and Handford 2014. Constructs were expressed in E. coli BL21 cells transformed with a pQE30 (Qiagen) based protein expression construct and a pREP4 plasmid for control of expression via the Lac Repressor.…”

Section: Methods and Experimentsmentioning

confidence: 99%

1H, 13C and 15N assignments of EGF domains 4 to 7 of human Notch-1

2014

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The Notch pathway is a core cell-cell signaling system in Metazoa which plays a key role in development and adult homeostasis. Whereas most Notch structural biology research has focused on the negative regulatory region and the intracellular domain, relatively little structural information is available for the extracellular part of human Notch-1 (hN-1) which mediates ligand recognition. This region consists of 36 epidermal growth factor-like (EGF) domains, many of which contain a calcium-binding consensus sequence. The calcium-binding site in each case is located within the N-terminal portion of the domain, and is associated with both intra- and inter-domain rigidity. The absence of calcium-binding sites in EGF6, EGF10 and EGF22, however, suggests that these domains might represent regions of flexibility in the receptor which could influence the cell-surface architecture (usually depicted as an extended rod projecting from the cell surface). To probe this, we have purified a quadruple-domain construct from hN-1, in which the non-calcium-binding EGF6 is flanked by EGF4-5 and EGF7. Here, we report (1)H, (13)C and (15)N resonance assignments for this four-domain 157 amino acid construct. The assignments presented here are the prerequisite for a detailed study of the structure and dynamics of this region of the Notch receptor.

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Bacterial Expression and In Vitro Refolding of Limited Fragments of the Notch Receptor and Its Ligands

Cited by 5 publications

References 14 publications

Non-Linear and Flexible Regions of the Human Notch1 Extracellular Domain Revealed by High-Resolution Structural Studies

Non-Linear and Flexible Regions of the Human Notch1 Extracellular Domain Revealed by High-Resolution Structural Studies

1H, 13C and 15N assignments of EGF domains 8–11 of human Notch-1

1H, 13C and 15N assignments of EGF domains 4 to 7 of human Notch-1

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