Bfl-1 HSQC-NMR Binding Assay

The Mcl-1 HSQC-NMR studies were performed as previously reported.^{17 (link),19 (link)} HSQC NMR experiments with Bfl-1 proceeded as follows, NMR spectra were acquired in NMR Buffer (20 mM NaH₂PO₄/Na₂HPO₄ pH 7.0, 150 mM NaCl, 0.5 mM DTT, and 0.5 mM TECP) supplemented with 10% D₂O and 4% DMSO using 5mm D₂O matched Shigemi NMR tubes. ¹⁵N labeled Bfl1-His (50 μM) was incubated with compounds 1 and 23. All NMR data were collected on a Bruker Avance NEO 600 MHz spectrometer at 298K with a TCl-H&F/C/N cryoprobe. A transverse relaxation optimized spectroscopy (TROSY) with a solvent suppression pulse sequence was used to acquire all HSQC data. NMRPipe^{66 (link)} and Sparky (Goddard and Kneller, Sparky 3, University of California, San Francisco) were used for all NMR data processing and analysis. Assignments of Bfl-1 residues were obtained from the printed HSQC as it was not deposited online by previous studies.^{67 (link)}