FACS enrichment and NMR-based structural determination and titrations with the α CTT peptides. (A) Representative images for the 10,000 yeast cells sorted on BD FACS Aria fusion cell sorter. Four rounds of sorting experiments were performed. The population P2, marked in blue (Q2 quadrant), is collected (>4,000 cells during each sorting). Q3 quadrant represents the unstained population. Expression of the binders in the SSO7D library was marked by labeling the c-myc tag in Alexa Fluor 633 channel (Q4 in first FACS and Q3 in second, third, and fourth FACS), and the binder-bound peptide was labeled with FITC (first FACS) or PE (second, third, and fourth FACS) channel (see Materials and methods). (B) The table represents the abundance (% enrichment) of the clones obtained after the fourth sorting after analyzing 10 single yeast colonies. S.No., serial number. (C) Sensogram obtained from the immobilized biotin-Hs_TUBA1A peptide on an SA surface in the SPR experiment to determine the binding parameters of the two abundant clones (A1aY1 and A1aY2) for their binding parameters. Binders (A1aY1 and A1aY2) were purified with GFP tag at the C terminus and were used at ∼8 µM concentrations to obtain the binding sensogram. RU, response unit. (D) 2D NMR spectra (HSQC spectra) and assignment of the peaks for corresponding amino acids in A1aY1 protein labeled with ¹⁵N and ¹³C isotopes. The x axis of the HSQC spectra represents the proton (¹H) shift and the y axis the ¹⁵N shift. (E) HSQC spectra of the ¹³C ¹⁵N–labeled A1aY1 protein in presence of α-tubulin CTT peptides (Hs_TUBA1A and Hs_TUBA1A-ΔY). A titration of A1aY1 protein (200 µM) with 1:0.5, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, and 1:7 excess of Hs_TUBA1A peptides showed a positive chemical shift in the residues of A1aY1, while titration with Hs_TUBA1A-ΔY in 1:1, 1:2, 1:3, and 1:4 excess did not show any significant change in the position of the residues. (F) Zoomed-in residue from the titration of Hs_TUBA1A and Hs_TUBA1A-ΔY (marked with black dashed box). (G) Values of the CSPs calculated from the ppm shift in the residues upon titration with Hs_TUBA1A. The protein sequence of the binder, A1aY1 with diversified residues (marked in red) are marked above the graph. (H) Left: The NMR structure of A1aY1 binder (magenta) showing the randomized residues (yellow) in SSO7D structure lying in the binding pocket for Hs_TUBA1A peptide. Right, A1aY1 binder colored according to surface charge; basic (blue), acidic (red), hydrophobic (yellow) and polar (green).