Figure 6

Overview of the RaPID selection procedure coupled to protein structure determination. See references^8,9 for more details. The starting DNA library is transcribed into an mRNA pool which is ligated with puromycin-derivatized oligonucleotides, then used as templates for in vitro translation. This translation reaction mixture contains 19 proteinogenic amino acids (excepting methionine) and is supplemented with an initiator tRNA acylated with chloroacetyl-d-tyrosine. The peptide is cyclised by intramolecular S_N2 reaction between the chloroacetyl group and a C-terminal cysteine. The puromycin covalently links the coding mRNA strand to the corresponding translated CPs, so reverse transcription generates mRNA/cDNA-fused CPs. The mixture is incubated with magnetic beads to select for CPs binding to the immobilized target, then washed, before the cDNA associated with bound CPs is PCR amplified and sequenced. The peptides thus identified are synthesized by standard solid-phase synthesis, incubated with the target protein, and tested by MS-based screening (or other binding assay) for complex formation (kinetic assays may also be performed). Co-crystallization experiments involve mixing CPs and target protein typically at a 1–1.1:1 molar ratio, respectively. Alternatively, the complex can be purified by size exclusion chromatography. RaPID screens could also be performed on enzyme–substrate/-inhibitor complexes to promote identification of non-competitive CPs (NCCPs). Owing to their typically high stability, CPs are well-tolerated for screening a broad range of crystallization conditions with extremes of pH, temperature, ionic strength, ligands/additives, and precipitants. Thus, crystallization conditions screened are limited by the stability of the target protein, not that of the CP crystallization agent, which is often an issue with antibodies/‘crystallization chaperones’⁵.