Fig. 1: Yeast-encoded macrocyclic peptide libraries.

a Schematic representation of the yeast display macrocyclic peptide (MP) system developed in this work. The cysteine-rich peptide sequence of interest (blue) is expressed as N-terminal fusion of a cysteine-free glycosylphosphatidylinositol cell-surface anchor protein (black line). A linker is placed between the peptide and the hemagglutinin (HA) tag (red). Bridging of one pair of cysteines yields MPs with ‘one ring’ and a unique topology, bridging two pairs of cysteines yields MPs with ‘two rings’ (three different topologies). b Five naïve libraries were generated to include either two or three cysteines (C, blue) at fixed positions and between 7 and 12 random amino acids (X). Naïve libraries were numbered as follows: 1 (CX₇C), 2 (CX₉C), 3 (CX₃CX₉C), 4 (CX₆CX₆C), and 5 (CX₉CX₃C). The size of each library (unique peptide sequences) is reported and was determined as described in other works²². c Yeast-displayed MP naïve libraries were constructed using degenerated primers that allow all 20 amino acids in the randomised positions (X = ‘NNK’) and homologous recombination-based methods²². Heat map indicating the frequency of cysteines (d) and the ‘TAG’ amber stop codon (‘Z’) (e) in each library. Frequencies were determined by NGS analysis. The intensity of the colour correlates with the frequency of a given cysteine or ‘TAG’ stop codon in the peptide sequence. High and low frequency values are shown in dark and light colours, respectively. f Heat map of experimental frequency of each individual amino acid determined by NGS compared to the theoretical probability expected values for ‘NNK’ codon. Individual amino acids are indicated by a one-letter code. The intensity of the colour correlates with the frequency ratio. Enriched amino acids in naïve libraries are shown in dark blue, whereas depleted amino acids are shown in dark red. Amino acids with an experimental frequency/theoretical probability ratio of one are shown in white. g Frequency of the occurrence and distribution of additional cysteine in library 3. The three constant cysteines are shown in blue, while the fourth random is highlighted in red. Data for d–g are provided as Source data.