Fig. 6.

Second generation TEAD binder has favorable stability. a Reversed-phase (RP) HPLC trace of TB1G2 under non-reducing or strongly reducing (10 mM DTT) conditions (top). The sample under reducing conditions was analyzed in an in-line LC/MS mass spectrometer, identifying peaks of interest (middle). Peptide m/z of representative peak P1 (bottom) shown, corresponding to a mass of 4971.4 Da. The non-reduced peptide’s mass was measured at 4968.7 Da on the same instrument. Full mass spectra available in Supplementary Fig. 10. b RP-HPLC of TB1G1 and TB1G2 under either non-reducing (NR) or intracellular reducing conditions using 10 mM glutathione (GSH). c 293F cells expressing SDGF-TB1G1 (top) or SDGF-TB1G2 (bottom) were incubated with either PBS, 10 mM glutathione (GSH) or 10 mM DTT for 5 mins before being washed and tested for TEAD binding (20 nM, 2-step stain with Alexa Fluor 647-streptavidin). d A control peptide, with known sensitivity to proteases, was cloned into SDPR and incubated with PBS or either 40 µg mL⁻¹ trypsin (top left) or 40 µg mL⁻¹ chymotrypsin (top right), followed by treatment with reducing agent (5 mM DTT) and iFluor 647 anti-6xHis staining. Bottom: Same as top, with cells expressing SDPR-TB1G2. e, f Circular dichroism spectra of soluble CDPs TB1G2 e and TB1G2-W40P f indicate a structure dominated by α-helical elements, and that this secondary structure signature is identical before (Pre) and after (Post) incubation at 95 °C. Insets: relative ellipticity at 220 nm during heating from 20 °C to 95 °C. g SYPRO Orange thermal shift assay of optides. Shown is the slope of the change in relative fluorescence units (dRFU dtemp⁻¹) during heating from 20 °C to 95 °C. Human siderocalin (HuScn) produced an expected melting temperature of 79 °C, as interpreted by the peak of its RFU vs temperature slope. Conversely, no melting temperature could be determined for the two optides tested (TB1G2 and TB1G2-W40P)