Fig. 2: l-monobody selection and characterization against d-Bcr-Abl SH2.

a Amino acid sequences of d-Abl SH2 l-monobody binders (DAM) generated by phage and yeast display selection based on the combinatorial ‘side-and-loop’ library. In the library designs, “X” denotes a mixture of 30% Tyr, 15% Ser, 10% Gly, 5% Phe, 5% Trp, and 2.5% each of all the other amino acids except for Cys; “O” denotes a mixture of Asn, Asp, His, Ile, Leu, Phe, Tyr, and Val; “U” denotes a mixture of His, Leu, Phe, and Tyr; and “Z” denotes a mixture of Ala, Glu, Lys, and Thr. A hyphen indicates a deletion. b Binding titrations in the yeast surface display format to estimate binding affinities of l-monobodies to d-Abl SH2. The mean fluorescence intensities of yeast cells displaying a monobody are plotted as a function of the concentration of the target and fitted to a 1:1 binding model. c Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis (performed once), (d) retention volumes in size exclusion chromatography (SEC) purification and expression yields, and (e) SEC chromatograms of selected l-DAMs recombinantly expressed in E. coli. (f) Competitive fluorescence polarization (FP) assay of l-DAMs incubated with d-Abl SH2 and fluorescently labeled d-pYEEI peptide binders in comparison with previously published l-monobodies HA4 (pY peptide competitor) and AS25 (allosteric binder) against l-Abl SH2 incubated with the corresponding l-pYEEI peptide. Measured data from two independent experiments (depicted as dots) were averaged. g–j Isothermal titration calorimetry (ITC) measurements of recombinant l-monobodies (g) DAM21 and (h) DAM27 titrated to the synthetic d-Abl SH2 domain. Each panel shows the raw heat signal of an ITC experiment (top) and the integrated calorimetric data of the area of each peak (bottom). The continuous line represents the best fit of the data based on a 1:1 binding model computed from the MicroCal software. Binding parameters including K_d value, stoichiometry (N), enthalpy (∆H), free enthalpy (∆G) and −T∆S calculated from the fit of each experiment are shown in (i) and (j). A representative measurement of at least two ITC experiments for each monobody is shown. Source data of (a, c and f) are provided as a Source Data file.