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. Author manuscript; available in PMC: 2020 Jul 31.
Published in final edited form as: J Am Chem Soc. 2019 Jul 5;141(30):11912–11922. doi: 10.1021/jacs.9b02963

Figure 3.

Figure 3.

Conformation-selective versions of TCO-conjugated probes. (A) Schematic for conversion of 2 and 3 (left) into 2-TCO and 3-TCO (right), respectively. (B) Inhibition curves and Ki values of 2, 2-TCO, 3, and 3-TCO against recombinant Src V284C activity. Values shown are mean ± SEM, n = 3. (C) 1-TCO, 2-TCO and 3-TCO promote expected levels of intermolecular SH3 domain accessibility of Src V284C. Schematic of how conformation-selective probes are predicted to modulate the intramolecular regulatory interactions and global confomrational state of Src (left). Percentage retained Src in an SH3 pulldown assay with recombinant Src V284C treated with 1-TCO, 2-TCO or 3-TCO (right). Values shown are mean ± SEM, n = 3. (D) 2-TCO and 3-TCO selectively enrich Src V284C from cell lysates. Cell lysates containing either Src V284C or Src WT were treated with 2-TCO or 3-TCO (5 μM), incubated with Tz-beads, and captured proteins were eluted under reducing and denaturing conditions. A Src immunoblot of the flow through (FT) and elution (EL) are shown. (E) 2-TCO–but not 3-TCO–selectively enriches Src V284C from cells. HEK293 cells expressing either Src V284C or Src WT were treated with the indicated concentrations of 2-TCO or 3-TCO, lysed, and then incubated with Tz-beads. Captured proteins were eluted under reducing and denaturing conditions. A Src immunoblot of the flow through (FT) and elution (EL) are shown. (F) 1-TCO and 2-TCO selectively enrich Src V284C from HeLa cells. HeLa cells expressing either Src V284C or Src WT were treated with 1-TCO (5 μM) or 2-TCO (2.5 μM), lysed, and then incubated with Tz-beads. Captured proteins were eluted under reducing and denaturing conditions. A Src immunoblot (left) and qunatification (right) of the flow through (FT) and elution (EL) are shown. (G) Kinetics of probe-bound target capture with Tz-beads. HeLa cells expressing either Src V284C or Src WT were treated with 2-TCO (2.5 μM), lysed, and then incubated with Tz-beads at 4 °C for the reaction time indicated. Immunoblots of the total lysate input (IN) and eluted proteins (EL) are shown.