inverse nmr methods  (Biofluids Inc)

Journal: The Journal of Biological Chemistry

Article Title: Dimeric Switch of Hakai-truncated Monomers during Substrate Recognition

doi: 10.1074/jbc.M114.592840

Figure Lengend Snippet: NMR structure of HYBΔC (aa 106–194). A, superposition of 20 energy-minimized conformers representing the NMR structure of HYBΔC. B, ribbon diagram of the lowest-energy conformer representing the three-dimensional NMR structure of HYBΔC. Each monomer contains three zinc-coordination sites: Sites 1, 2, and 3. The zinc ions are shown as red spheres. C, metal coordinations in the NMR structure of HYBΔC. The coordination of zinc ions in the RING domain and the C-terminal domain of HYBΔC are shown.

Article Snippet: The 15N relaxation times, T1 and T1ρ, were measured at 25 °C using inversely detected two-dimensional NMR methods (31, 32) on a Bruker 800 Avance machine equipped with a TXI cryogenic probe.

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: Dimeric Switch of Hakai-truncated Monomers during Substrate Recognition

doi: 10.1074/jbc.M114.592840

Figure Lengend Snippet: ITC, gel filtration profiles, AUC, and NMR relaxation studies of HYBΔC with phospho-E-cadherin-(747–759). A, the tyrosine-phosphorylated E-cadherin-(747–759) was titrated against HYBΔC using ITC. The top panel shows the heat release profile after baseline correction and the lower panel indicates the binding isotherm for the interaction. The dissociation constant (Kd) and binding stoichiometry (N) are shown in the table. B, comparison of gel filtration profiles of HYBΔC in the presence (blue) and absence (red) of phospho-E-cadherin-(747–759) using a Superdex 75 gel filtration column. The elution profile suggests that HYBΔC exists as a dimer in the presence of ligand but as a monomer in the absence of the ligand. C, AUC analysis of HYBΔC in the presence of phospho-E-cadherin-(747–759) ligand. The ligand-induced dimerization of HYBΔC was studied using sedimentation velocity analysis. The results show that the protein exists as a dimer in the presence of the ligand with an apparent molecular mass of 20,000 Da. D, 15N relaxation T1 (a) and T2 (b) values as well as the error values for each residues in free HYBΔC sample (labeled as a red star) and phospho-E-cadherin747–759 bound complex (labeled as ●). The results show the T1 and T2 values change in the presence and absence of the substrate peptide. The secondary structures and domain boundary of HYBΔC also are illustrated in the middle of the figure. The missing residues and weak intensity residues (except Val128) are located in the region between β3 and α2.

Article Snippet: The 15N relaxation times, T1 and T1ρ, were measured at 25 °C using inversely detected two-dimensional NMR methods (31, 32) on a Bruker 800 Avance machine equipped with a TXI cryogenic probe.

Techniques: Filtration, Binding Assay, Comparison, Sedimentation, Labeling