Weak pulling forces exerted on N_in‐orientated transmembrane segments during co‐translational insertion into the inner membrane of <i>Escherichia coli</i>

Transmembrane helices (TMHs) in membrane proteins can be orientated with their N‐terminus towards the cytoplasm (N in ), or facing the non‐cytoplasmic side (N out ). Most membrane proteins are inserted co‐translationally into membranes, aided by Sec‐type translocons. Since the final orientation of N in ‐ and N out ‐orientated TMHs differs, they could also interact differently with the translocon and the surrounding membrane during insertion. We measured pulling forces exerted on N in ‐orientated TMHs during co‐translational insertion into the inner membrane of Escherichia coli . Our results demonstrate that N in ‐orientated TMHs experience a weaker pulling force but retain the overall biphasic force profile seen previously for N out ‐orientated TMHs (Ismail et al., 2012 [1]).