(A) Measuring TM insertion using a glycosylation assay. Glycosylation sites are engineered into the protein flanking the TM of interest. Glycosylation cannot occur on one of the sites if the TM has inserted across the membrane. The amount of single and doubly glycosylated protein is compared with calculate the ΔG_app of membrane insertion of that particular TM helix. (B) Measuring TM insertion using force profile analysis. In these experiments an arrest peptide (AP) is engineered into the protein of interest (B, i) to study the force generated by a nascent chain during translation in vivo [12,74,75]. APs stall translation with a duration that is proportional to the force exerted on the nascent chain during translation by the ribosome. Where little force is generated on the nascent chain (e.g. L₁, where TM2 is not interacting with SecYEG, B, ii), the AP stalls translation and only arrested products are generated. Where a higher force is generated (e.g. L₂, where TM2 is integrated into the membrane by SecYEG, B, iii), the AP stalling is released and a full-length product is generated. Mutations in a TM to introduce more hydrophilic residues (B, iv) can alter the force exerted on the nascent chain (TM2, green, compared with TM2^mut, purple). A plot of the fraction of full-length product against the position of the AP in the nascent chain amino acid sequence (L) provides details into the force acting on a nascent chain during translation (B, v).