ocrl rhogap domain Search Results


ash-rhogap domain of ocrl (residues 536–901 of human ocrl)  (Promega)
90
Promega ash-rhogap domain of ocrl (residues 536–901 of human ocrl)
Domain structures of INPP5B and <t>OCRL</t> and their F&H motif-containing interactors, and the crystal structure of the <t>human</t> <t>ASH-RhoGAP</t> domain in complex with the F&H peptide from human Ses1. (a) The ASH domain is colored in red, the RhoGAP domain in blue, and the Ses1 F&H peptide in yellow. Molecular graphics for this and all subsequent figures were generated with Pymol . (b) Close-up view of the interface between the F&H peptide (yellow) and the RhoGAP domain (blue). (c) F&H peptide sequences used in this study. Phosphoserine residues are indicated in green. (d) GST-ASH-RhoGAP constructs of OCRL were assayed for binding to APPL1 from rat brain homogenate. The GST negative control is marked as a -, and the OCRL constructs are either wild-type (WT), or mutated in the F&H binding site (W739A and D743R). As predicted, the mutants disrupt binding to APPL1, without affecting binding to clathrin heavy chain (CHC).
Ash Rhogap Domain Of Ocrl (Residues 536–901 Of Human Ocrl), supplied by Promega, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/ash-rhogap domain of ocrl (residues 536–901 of human ocrl)/product/Promega
Average 90 stars, based on 1 article reviews
ash-rhogap domain of ocrl (residues 536–901 of human ocrl) - by Bioz Stars, 2026-03
90/100 stars
  Buy from Supplier

Image Search Results


Domain structures of INPP5B and OCRL and their F&H motif-containing interactors, and the crystal structure of the human ASH-RhoGAP domain in complex with the F&H peptide from human Ses1. (a) The ASH domain is colored in red, the RhoGAP domain in blue, and the Ses1 F&H peptide in yellow. Molecular graphics for this and all subsequent figures were generated with Pymol . (b) Close-up view of the interface between the F&H peptide (yellow) and the RhoGAP domain (blue). (c) F&H peptide sequences used in this study. Phosphoserine residues are indicated in green. (d) GST-ASH-RhoGAP constructs of OCRL were assayed for binding to APPL1 from rat brain homogenate. The GST negative control is marked as a -, and the OCRL constructs are either wild-type (WT), or mutated in the F&H binding site (W739A and D743R). As predicted, the mutants disrupt binding to APPL1, without affecting binding to clathrin heavy chain (CHC).

Journal: Nature structural & molecular biology

Article Title: Recognition of the F&H motif by the Lowe Syndrome protein OCRL

doi: 10.1038/nsmb.2071

Figure Lengend Snippet: Domain structures of INPP5B and OCRL and their F&H motif-containing interactors, and the crystal structure of the human ASH-RhoGAP domain in complex with the F&H peptide from human Ses1. (a) The ASH domain is colored in red, the RhoGAP domain in blue, and the Ses1 F&H peptide in yellow. Molecular graphics for this and all subsequent figures were generated with Pymol . (b) Close-up view of the interface between the F&H peptide (yellow) and the RhoGAP domain (blue). (c) F&H peptide sequences used in this study. Phosphoserine residues are indicated in green. (d) GST-ASH-RhoGAP constructs of OCRL were assayed for binding to APPL1 from rat brain homogenate. The GST negative control is marked as a -, and the OCRL constructs are either wild-type (WT), or mutated in the F&H binding site (W739A and D743R). As predicted, the mutants disrupt binding to APPL1, without affecting binding to clathrin heavy chain (CHC).

Article Snippet: The ASH-RhoGAP domain of OCRL (residues 536–901 of human OCRL) was expressed in BL21 DE3 pRLIP (Promega) cells as a GST-fusion (pGex6P1, GE Healthcare Lifesciences) and purified with glutathione sepharose resin (GE Healthcare Lifesciences) in 20 mM Tris pH 8, 100 mM NaCl, 10% glycerol, 1 mM EDTA, and 1 mM DTT.

Techniques: Generated, Construct, Binding Assay, Negative Control

Patient mutations affecting F&H binding are global folding mutations. Patient mutations are represented in all structures as spheres. (a) The mapping of the patient missense mutations onto the ASH-RhoGAP structure, with two mutation networks expanded, one in the ASH domain and the other in the RhoGAP domain. (b) F&H binding is disrupted due to destabilization of OCRL. Inspection of a coomassie-stained gel shows significant degradation as well as the co-purification of a DnaK chaperone for mutants that display a lack of F&H binding. This destabilization is not seen in our designed F&H-binding mutation (W739A), a Rab5-defective mutant (F668V), or a splice-site mutation that results in a lack of expressed protein (A861T). Patient mutations which give rise to Dent 2 disease are indicated by an asterisk. (c) The GST-ASH-RhoGAP OCRL constructs in panel b were assessed for APPL1 binding using a GST pulldown assay from rat brain followed by western blot for APPL1.

Journal: Nature structural & molecular biology

Article Title: Recognition of the F&H motif by the Lowe Syndrome protein OCRL

doi: 10.1038/nsmb.2071

Figure Lengend Snippet: Patient mutations affecting F&H binding are global folding mutations. Patient mutations are represented in all structures as spheres. (a) The mapping of the patient missense mutations onto the ASH-RhoGAP structure, with two mutation networks expanded, one in the ASH domain and the other in the RhoGAP domain. (b) F&H binding is disrupted due to destabilization of OCRL. Inspection of a coomassie-stained gel shows significant degradation as well as the co-purification of a DnaK chaperone for mutants that display a lack of F&H binding. This destabilization is not seen in our designed F&H-binding mutation (W739A), a Rab5-defective mutant (F668V), or a splice-site mutation that results in a lack of expressed protein (A861T). Patient mutations which give rise to Dent 2 disease are indicated by an asterisk. (c) The GST-ASH-RhoGAP OCRL constructs in panel b were assessed for APPL1 binding using a GST pulldown assay from rat brain followed by western blot for APPL1.

Article Snippet: The ASH-RhoGAP domain of OCRL (residues 536–901 of human OCRL) was expressed in BL21 DE3 pRLIP (Promega) cells as a GST-fusion (pGex6P1, GE Healthcare Lifesciences) and purified with glutathione sepharose resin (GE Healthcare Lifesciences) in 20 mM Tris pH 8, 100 mM NaCl, 10% glycerol, 1 mM EDTA, and 1 mM DTT.

Techniques: Binding Assay, Mutagenesis, Staining, Copurification, Construct, GST Pulldown Assay, Western Blot