HIV-1 particle formation and release occur with oligomerization of Gag polyprotein precursor and budding through the cellular plasma membrane. Maturation to an infectious virion depends on multiple proteolytic cleavages of the viral polyproteins by the viral protease, PR. PR is part of the Gag-Pro-Pol polyprotein, a minor frameshifted translational variant of the Gag protein that is incorporated in the budding virion with Gag. PR is active as a dimer and must exist both in an active form in the context of the Gag-Pro-Pol precursor and as the mature dimer. Here we study the cotranslational folding of the PR monomer within frameshifted transframe-protease-reverse transcriptase (TF-PR-RT) constructs by in vitro translation to explore early steps of PR folding and activation. We demonstrate cotranslational folding of ribosome-bound PR at its conserved α-helix near the C-terminus. The experimental design included constructs that were either released from the ribosome, or retained on the ribosome by a translational arrest peptide constraining the PR domain to a monomeric state. Unexpectedly, we find that released TF-PR-RT dimers are refractory to cleavage by PR, while ribosome-bound monomeric chains are efficiently cleaved. We suggest that the “constrained isolation” of PR monomers on the ribosome in this system is analogous to PR monomers entering the budding virion in the context of the Gag-Pro-Pol precursor. These observations suggest a model for virion maturation in which dimerization of a subset of Pro-Pol precursors initiates cleavage of PR monomers that then dimerize and carry out most of the proteolytic processing needed for virion maturation.

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. 

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.

During SecYEG-mediated cotranslational insertion of membrane proteins, transmembrane helices (TMHs) first make contact with the membrane when their N-terminal end is ~ 45 residues away from the peptidyl transferase centre. However, we recently uncovered instances where the first contact is delayed by up to ~ 10 residues. Here, we recapitulate these effects using a model TMH fused to two short segments from the Escherichia coli inner membrane protein BtuC: a positively charged loop and a re-entrant loop. We show that the critical residues are two Arg residues in the positively charged loop and four hydrophobic residues in the re-entrant loop. Thus, both electrostatic and hydrophobic interactions involving sequence elements that are not part of a TMH can impact the way the latter behaves during membrane insertion. 

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.

Fluorescence assay technologies are commonly used in high-throughput screening because of their sensitivity and ease of use. Different technologies have their characteristics and the rationale for choosing one over the other can differ between projects because of factors such as availability of reagents, assay performance, and cost. Another important factor to consider is the assay susceptibility to artifacts, which is almost as important as the ability of the assay to pick up active compounds. Spending time and money on false positives or missing the opportunity to build chemistry around false negatives is something that every drug project tries to avoid. We used a BET family Bromodomain, BRD4(1), to explore the outcome of a screening campaign using three fluorescent assay technologies as primary assays. A diverse 7,038 compound set was screened in fluorescence lifetime, fluorescence polarization, and homogeneous time-resolved fluorescence to look at primary hit rates, compound overlap, and hit confirmation rates. The results show a difference between the fluorescence assay technologies with three separate hit lists and some overlap. The confirmed hits from each assay were further evaluated for translation into cells (NanoBRET (TM)). Most of the actives confirmed in cells originated from compounds that overlapped between the assays. In addition, a well-annotated set of compounds with undesirable mechanism of inhibition was screened against BRD4(1) to compare the ability to discriminate true hits from artifact compounds. The results indicate a difference between the assays in their ability to generate false positives and negatives.