Fig. 1

Established workflow for identifying permissive stretches (PSs) in proteins and design of protein knockdowns. The established workflow is exemplified with adenylate kinase (Adk) and requires primary structure information alone. a Gaps in a multiple sequence alignment (MSA) of several (>5) homologous proteins indicate stretches in a protein likely permissive to insertion of additional amino acid residues. b The span of a PS is defined as the gap in the alignment plus its flanking residues. The four identified PSs within Adk are indicated with Roman numerals. c The design of protein knockdowns requires the insertion of a Tobacco etch virus protease recognition site (TEV-tag) into a flexible, surface accessible PS. Relative surface accessibility (RSA) and structural context of a PS can be predicted based on primary structure information. RSA values for each PS within Adk are indicated and were calculated by computing the geometric means of the RSA values of adjacent residue pairs within a given stretch and taking their maximum value. RSA values range from 0 (buried) to 1 (fully exposed). The average maximum geometric mean RSA of a random stretch was determined to be 0.30. For illustration, PSs were mapped onto the surface representation of the crystal structure of Adk (Protein Data Bank (PDB) 1AKE). d The information acquired above guides the identification of a potentially functional, surface exposed, and flexible PS for chromosomal TEV-tag insertion. PSII shows a high RSA, and secondary structure prediction indicates that it stretches across a 6-residue loop. PSIII shows the same RSA as PSII and it stretches across an 18-residue loop. But PSII was shown to be functionally relevant ([42] and Fig. 2) and therefore not chosen for TEV-tag insertion