Proteins are a diverse class of biomolecules that carry out many essential functions across all organisms. They can either be found in aqueous compartments or embedded in biological membranes of the cell and are called soluble or membrane proteins, respectively. Membrane proteins are involved in many essential cellular pathways and account for about one third of both pro- and eukaryotic proteomes. They are major drug targets and are critical when cells are engineered to secrete therapeutic proteins and industrial enzymes.

In order to be functional, proteins must fold into specific 3-dimensional structures, be targeted to the right destination and can undergo additional maturation steps. Most studies have focused on the characterisation of fully synthesized proteins, and much less is known about their biogenesis while still being translated by the ribosome. Here, we focus on cotranslational events studied in the well-characterised model bacterium Escherichia coli, and take advantage of a recently developed technology that uses so-called translational arrest peptides (APs). APs stall their own translation on the ribosome unless a sufficient pulling force is applied on the nascent polypeptide chain, and can therefore be used as molecular force sensors. We found that enough force to overcome AP-induced translational arrest can be generated by transmembrane helices (TMHs) as they insert into the E. coli inner membrane. By following the stepwise cotranslational insertion of three multi-spanning integral membrane proteins, we found that a TMH starts generating a force on the nascent chain when it reaches about 45 residues away from the ribosomal peptidyl transferase center (PTC). At this distance the TMH is expected to be in the vicinity of the bacterial SecYEG translocon and begin to insert into the lipid bilayer. Interestingly, this force can be affected by the presence of other membrane-interacting segments flanking the TMH. Another intriguing finding was that an N-terminal globular domain can fold well before the downstream membrane domain starts to integrate into the membrane. Furthermore, we detected forces that are generated by residue-specific intrachain as well as interchain interactions, which suggest cotranslational folding and oligomerisation of membrane proteins. Finally, we recorded the force that is generated by a recombinant soluble protein as it folds cotranslationally in E. coli. The onset of the folding was detected when the protein’s C-terminus has not yet fully emerged from the ribosome exit tunnel, and folding was delayed in the presence of the cotranslationally acting chaperone trigger factor (TF).

Taken together, the work presented in this thesis has led to a better understanding of how proteins fold, assemble as well as insert into a biological membrane during their translation, and has revealed multiple factors that contribute to the complexity of cotranslational protein biogenesis.