Human growth hormone (hGH) is a four-helix bundle protein of considerable pharmacological interest. Recombinant hGH is produced in bacteria, yet little is known about its folding during expression in Escherichia coli. We have studied the cotranslational folding of hGH using force profile analysis (FPA), both during in vitro translation in the absence and presence of the chaperone trigger factor (TF), and when expressed in E. coli. We find that the main folding transition starts before hGH is completely released from the ribosome, and that it can interact with TF and possibly other chaperones. 

In recent years, it has become clear that many homo- and heterodimeric cytoplasmic proteins in both prokaryotic and eukaryotic cells start to dimerize cotranslationally (i.e., while at least one of the two chains is still attached to the ribosome). Whether this is also possible for integral membrane proteins is, however, unknown. Here, we apply force profile analysis (FPA)—a method where a translational arrest peptide (AP) engineered into the polypeptide chain is used to detect force generated on the nascent chain during membrane insertion—to demonstrate cotranslational interactions between a fully membrane-inserted monomer and a nascent, ribosome-tethered monomer of the Escherichia coli inner membrane protein EmrE. Similar cotranslational interactions are also seen when the two monomers are fused into a single polypeptide. Further, we uncover an apparent intrachain interaction between E14 in transmembrane helix 1 (TMH1) and S64 in TMH3 that forms at a precise nascent chain length during cotranslational membrane insertion of an EmrE monomer. Like soluble proteins, inner membrane proteins thus appear to be able to both start to fold and start to dimerize during the cotranslational membrane insertion process. 

We follow the cotranslational biosynthesis of three multispanning Escherichia coli inner membrane proteins in vivo using high-resolution force profile analysis. The force profiles show that the nascent chain is subjected to rapidly varying pulling forces during translation and reveal unexpected complexities in the membrane integration process. We find that an N-terminal cytoplasmic domain can fold in the ribosome exit tunnel before membrane integration starts, that charged residues and membrane-interacting segments such as re-entrant loops and surface helices flanking a transmembrane helix (TMH) can advance or delay membrane integration, and that point mutations in an upstream TMH can affect the pulling forces generated by downstream TMHs in a highly position-dependent manner, suggestive of residue-specific interactions between TMHs during the integration process. Our results support the 'sliding' model of translocon-mediated membrane protein integration, in which hydrophobic segments are continually exposed to the lipid bilayer during their passage through the SecYEG translocon.