Cotranslational folding of the titin I27 domain by force-profile analysis. Cotranslational folding of the titin I27 domain by force-profile analysis. (A) The force-measurement assay. Modified with permission from ref. 2. I27, preceded by a His-tag, is placed L residues away from the last amino acid of the SecM AP, which in turn is followed by a 23-residue C-terminal tail derived from E. coli LepB. Constructs are translated for 15 min in the PURE in vitro translation system, and the relative amounts of arrested and full-length peptide chains produced are determined by SDS/PAGE. The fraction full-length protein, fFL, reflects the force exerted on the AP by the folding of I27 at linker length L. At short linker lengths (Top) there is not enough room in the exit tunnel for I27 to fold, little force is exerted on the AP, and the ribosome stalls efficiently on the AP (fFL ≈ 0). At intermediate linker lengths (Middle) there is enough room for I27 to fold but only if the linker segment is stretched, force is exerted on the AP, and stalling is reduced (fFL > 0). At long linker lengths (Bottom) I27 has already folded when the ribosome reaches the last codon in the AP, and again little force is exerted on the AP (fFL ≈ 0). (B) Force profiles for the I27 domain (solid squares) and the nonfolding (nf) mutant I27[W34E] (open squares). The standard error of fFL is calculated for values of L where three or more experiments were performed. Pengfei Tian et al. PNAS 2018;115:48:E11284-E11293 ©2018 by National Academy of Sciences