Journal: The Journal of Biological Chemistry

Article Title: Characterization of the binding mode of JNK-interacting protein 1 (JIP1) to kinesin-light chain 1 (KLC1)

doi: 10.1074/jbc.RA118.003916

Figure Lengend Snippet: Thermodynamic parameters of the ITC experiments between various KLC1-TPR fragments and mutants and JIP1-C10 peptides

Article Snippet: For experiments, the labeled sample was then adjusted to 40 n m with the ITC buffer containing 25 m m Hepes, pH 7.0, and 150 m m NaCl supplemented with 0.05% Tween 20 (NanoTemper Technologies).

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: Characterization of the binding mode of JNK-interacting protein 1 (JIP1) to kinesin-light chain 1 (KLC1)

doi: 10.1074/jbc.RA118.003916

Figure Lengend Snippet: Mechanism of JIP1-Cter binding to KLC1-TPR domain. A, isothermal titration calorimetry measurements of the reference KLC1-TPR-[A1-B6] fragment with the JIP1-C10-wt peptide. This reference measurement was performed at 25 °C in a buffer containing 25 mm Hepes, pH 7.0, and 150 mm NaCl. B, superposition of ITC curves of the reference KLC1-TPR-[A1-B6] fragment with the JIP1-C10-wt peptide at 25 °C in a buffer containing variable ionic strength (150, 250, or 500 mm NaCl). C, superposition of ITC curves showing the interaction between the reference KLC1-TPR-[A1-B6] fragment and JIP1-C10-E706A/D707A double mutant (blue), JIP1-C10-I708A/L710A double mutant (green), and JIP1-C10-Y709A mutant (red). For comparison, the reference measurement between the KLC1-TPR-[A1-B6] fragment and the JIP1-C10-wt peptide is reported (black). Sequence alignment of the different JIP1-C10 peptides used for this experiment is shown below. The solid lines drawn through the data points match the best fit to the data.

Article Snippet: For experiments, the labeled sample was then adjusted to 40 n m with the ITC buffer containing 25 m m Hepes, pH 7.0, and 150 m m NaCl supplemented with 0.05% Tween 20 (NanoTemper Technologies).

Techniques: Binding Assay, Isothermal Titration Calorimetry, Mutagenesis, Comparison, Sequencing

Journal: The Journal of Biological Chemistry

Article Title: Characterization of the binding mode of JNK-interacting protein 1 (JIP1) to kinesin-light chain 1 (KLC1)

doi: 10.1074/jbc.RA118.003916

Figure Lengend Snippet: Binding experiments between SH2D6 peptides and the reference KLC1-TPR-[A1-B6] fragment. A, sequence alignment of the different SH2D6 and JIP1-C10 peptides used for these binding experiments. B, superposition of ITC curves showing the interaction between the reference KLC1-TPR-[A1-B6] fragment and JIP1-C10 WT (reference measurement; black), SH2D6-[172–181] peptide (blue) and SH2D6-[170–181] peptide (green), as well as the interaction between an equivalent KLC2-TPR-[A1-B6]-wt fragment and SH2D6-[170–181] peptide (red). The solid lines drawn through the data points match the best fit to the data. C, superposition of MST curves showing the interaction between the labeled KLC1-TPR-[A1-B6] fragment and JIP1-C10 WT (reference measurement; green) and the SH2D6-[172–181] (red) peptides. The curves represent an average of three independent measurements with the S.D. shown by error bars.

Article Snippet: For experiments, the labeled sample was then adjusted to 40 n m with the ITC buffer containing 25 m m Hepes, pH 7.0, and 150 m m NaCl supplemented with 0.05% Tween 20 (NanoTemper Technologies).

Techniques: Binding Assay, Sequencing, Labeling

Journal: The Journal of Biological Chemistry

Article Title: Characterization of the binding mode of JNK-interacting protein 1 (JIP1) to kinesin-light chain 1 (KLC1)

doi: 10.1074/jbc.RA118.003916

Figure Lengend Snippet: Minimal region of KLC1-TPR for JIP1-C10 binding. Superposition of ITC curves shows the interaction between the JIP1-C10-wt peptide and N-terminal truncated fragments (A) and C-terminal truncated fragments (B) of KLC1-TPR domain. For comparison, the reference measurement between the JIP1-C10-wt peptide and the complete KLC1-TPR-[A1-B6] fragment is reported (black). C, color-based representation of the 3D template structure of the KLC1-TPR domain (PDB code 3NF1) according to the -fold decrease in binding affinity (Kd) measured in ITC. The solid lines drawn through the data points match the best fit to the data.

Article Snippet: For experiments, the labeled sample was then adjusted to 40 n m with the ITC buffer containing 25 m m Hepes, pH 7.0, and 150 m m NaCl supplemented with 0.05% Tween 20 (NanoTemper Technologies).

Techniques: Binding Assay, Comparison

Journal: The Journal of Biological Chemistry

Article Title: Characterization of the binding mode of JNK-interacting protein 1 (JIP1) to kinesin-light chain 1 (KLC1)

doi: 10.1074/jbc.RA118.003916

Figure Lengend Snippet: Identification of critical residues of KLC1-TPR for JIP1-C10 binding. A, KLC1-TPR residues examined by ITC for their binding to JIP1-C10-wt peptide are indicated by white spheres and labeled on the 3D template structure of the KLC1-TPR domain. The critical Asn343 position is indicated with a red star (15). KLC1-TPR is shown in a cartoon and colored according to the -fold decrease in binding affinity (Kd) measured in ITC, as shown in Fig. 4C. B and C, superposition of ITC curves showing the interaction of the JIP1-C10-wt peptide with the asparagine mutants (B) and the arginine/lysine mutants (C) of the reference KLC1-TPR-[A1-B6] fragment. For comparison, the reference measurement between the JIP1-C10-wt peptide and the complete KLC1-TPR-[A1-B6] fragment is reported (black). The solid lines drawn through the data points match the best fit to the data. D, surface representation of the 3D structure of KLC1-TPR template (PDB code 3NF1; same orientation as in A) colored in white with critical residues indicated in red and residues in close proximity indicated in pink.

Article Snippet: For experiments, the labeled sample was then adjusted to 40 n m with the ITC buffer containing 25 m m Hepes, pH 7.0, and 150 m m NaCl supplemented with 0.05% Tween 20 (NanoTemper Technologies).

Techniques: Binding Assay, Labeling, Comparison

Journal: bioRxiv

Article Title: The RNA chaperone protein CspA stimulates translation during cold acclimation by promoting the progression of the ribosomes

doi: 10.1101/2021.05.24.445485

Figure Lengend Snippet: Titration of E. coli ribosomal particles with CspA was studied at 25°C (A-E) and 35 °C (F-H). Titration was carried out by consecutive 2 µL injections of CspA in a cell containing 200 µL of either 70S ribosomes (A, and F), 30S subunits (B and G) or 50S subunits (C and H). D and E are examples of signals of CspA and E. coli 50S subunits obtained upon dilution in ITC buffer at 25 °C: in (D) 2 µL of CspA were repeatedly injected in the sample cell filled with ITC buffer, while in (E) 2 µL of ITC buffer were repetitively injected in the sample cell filled with 50S subunits.

Article Snippet: All samples were dialyzed against ITC buffer using centrifugal filter units (Centricon, Merck Millipore), 3 K for CspA and 100 K for E. coli ribosome.

Techniques: Titration, Injection