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. 2023 May 12;14:2745. doi: 10.1038/s41467-023-38220-2

Fig. 2. 2H influenced the biochemical activities of Msi3 in different ways.

Fig. 2

a The ATP-induced allosteric coupling assayed by limited trypsin digest. control: trypsin was not added. Apo: nucleotide was not added. +ATP: ATP was included at a final concentration of 2 mM. b The effect of 2H on the peptide substrate binding activity of Msi3. A fluorescent polarization assay was carried out using the fluorescently labeled TRP2-181 peptide as a substrate. Top: binding curves with dissociation constants (Kd) listed in parentheses. The polarization value is an indication of Msi3 binding to the TRP2-181 peptide. Data are presented as mean values + /- SEM (n = 3 independent experiments). Bottom: competition assay with 2H. Msi3 was first incubated with the TRP2-181 peptide to allow binding, and then 2H was added to compete off the pre-bound TRP2-181 peptide. Polarization values were monitored over time, with the concentrations of Msi3 labeled. Reactions without 2H were used as controls. buffer: the TRP2-181 peptide only. c NEF activity of Msi3 on Ssa1. After a complex formed between Ssa1 and ATP-FAM, the reduction of polarization values represented the release of ATP-FAM from Ssa1 upon the addition of ATP or/and Msi3. Release kinetics (koff) are summarized in Fig. S5. d Formation of the Msi3-Ssa1 complex. Complex formation was analyzed using a native polyacrylamide gel electrophoresis (PAGE), then visualized after staining with Coomassie blue. For all assays, 2H was added at a concentration of 100 µM unless labeled otherwise. Source data are provided as a Source Data file for all the panels.