Biaevaluation software 2

The binding between Tpm and SMTNL1-TMB with or without phosphorylation was evaluated by SPR using a BIAcore X100 instrument (GE Healthcare). Purified Tpm was immobilized via amine-coupling onto a CM5 sensor chip (GE Healthcare). The running buffer contained 20 mM HEPES pH 7.5, 100 mM KCl, 1 mM DTT, and 0.005% (v/v) Tween-20. Five concentrations of SMTNL1-TMB from 0.12 to 10 μM were prepared by serial dilution and were injected at a flow rate of 30 μL/min with a contact time of 1 min at 25 °C. The chip was regenerated by injecting 1 M NaCl for 4 min followed by glycine-HCl (10 mM, pH 3.0) for 1 min. Each experiment was repeated three times (n = 3) to obtain a standard error (SE). The BIAevaluation software 2.0 (GE Healthcare) was used to analyze the SPR sensorgrams and to obtain the dissociation constants (Kd).

The binding between Ca²⁺-bound LPL-EF and a synthetic peptide corresponding to H5 (Ac-STDVAKTFRKAINKKEGI-NH2) was evaluated by SPR using a BIAcore X100 instrument (GE Healthcare) and was compared to other peptides including the cytotoxic peptide melittin, and the CaM-target CaMKI and smMLCK peptides. Peptides used here were purchased as synthetic peptides with >95% purity from Genscript (San Diego, CA). The EF construct was immobilized via its sole cysteine residue (Cys42) onto a CM5 sensor chip (GE Healthcare) using thiol-coupling. The running buffer contained 10 mM Tris-HCl pH 7.5, 150 mM KCl, 1 mM CaCl₂, and 0.005% (v/v) Tween-20. Different concentrations of the peptide sample were injected at a flow rate of 30 μL/min with a contact time of 1 min at 25 °C. The BIAevaluation software 2.0 (GE Healthcare) was used to process the SPR sensorgrams and for curve-fitting to obtain the dissociation constants (K_d’s). Two different concentrations in each experiment were injected twice to obtain the fitting errors (SEM).

The binding between holo-NosJ and other proteins was evaluated by SPR using a Biacore S200 instrument (GE Healthcare). For these experiments, holo-NosJ containing an N-terminal hexa-His tag was covalently immobilized onto the CM5 sensor chip (GE Healthcare) via amine coupling, and the analyses were performed by running different protein solutions over the chip surface. Holo-NosJ was expressed and purified from E. coli BAP1 as described in the Supplementary Methods and the tested proteins was estimated to be of >95% purity by SDS-polyacrylamide gel electrophoresis. The running buffer containing 10 mM Tris-HCl (pH 8.0), 100 mM NaCl, 3 mM EDTA and 0.005% (v/v) Tween-20. Proteins were injected at a flow rate of 30 μl min⁻¹ with a contact time of 1 min at 25 °C. The BIAevaluation software 2.0 (GE Healthcare) was used to process the SPR sensorgrams and for curve-fitting to obtain the dissociation constants (K_D) using a 1:1 interaction model.