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.

In vivo, the majority of nascent protein chains begin folding during translation in order to reach their native structure. While the importance of co-translational folding has become increasingly clear, the specific mechanisms underlying the coordination between the ribosome, the nascent chain and interacting partners are still uncertain. Here, we show that calmodulin (CaM) plays a prominent role at discrete steps of the co-translational folding pathway of the calcium responsive domain (CRD) of the human neuronal KV7.2 ion channel, providing grounds for the proposal of a likely folding pathway. By combining force profile analysis and single-molecule force spectroscopy techniques, we found that CaM, in a calcium-dependent manner, affects early folding events involving three key α-helices in the CRD. In addition, this study suggests that CaM at early stages participates in the formation of metastable helical hairpins, as part of the co-translational folding pathway. These findings expand on the role of CaM as a key regulator of co-translational folding.

HIV-1 particle assembly depends critically on multiple proteolytic cleavages of viral polyproteins by the viral protease, PR. PR is translated as part of the Gag-Pro-Pol polyprotein, which undergoes autoproteolysis to liberate active, dimeric PR during virus particle maturation. Gag-Pro-Pol is produced via an infrequent -1 frameshifting event in ribosomes translating full length genomic RNA as Gag mRNA. Here, we study the cotranslational folding and autoproteolytic processing of frameshifted transframe-protease-reverse transcriptase (TF-PR-RT) constructs by in vitro translation. We demonstrate partial cotranslational folding of ribosome-bound PR at its conserved α-helix near the C terminus. Unexpectedly, we find that the initial dimerization of TF-PR-RT involves ribosome-bound nascent chains that are then not further cleaved. Moreover, only ribosome-bound nascent chains are substrates for PR-catalyzed processing. These observations are consistent with a model for virion assembly in which dimerization of a subset of Pro-Pol precursors leads to cleavage of PR monomers that then carry out the bulk of the proteolytic processing needed for virion maturation and infectivity.

Voltage-sensor domains (VSDs), found in many voltage-sensitive ion channels and enzymes, are composed of four transmembrane helices (TMHs), including the atypical, highly positively charged S4 helix. VSDs are cotranslationally inserted into the membrane, raising the question of how the highly charged S4 helix is integrated into the lipid bilayer as it exits the ribosome. Here, we have used force profile analysis (FPA) to follow the cotranslational insertion of the six-TMH KvAP voltage-sensitive ion channel into the Escherichia coli inner membrane. We find that the insertion process proceeds through three semi-independent steps: i) insertion of the S1-S2 helix hairpin, ii) insertion of the S3-S5 helices, and iii) insertion of the Pore and S6 helices. Our analysis highlights the importance of the concerted insertion of helical hairpins, the dramatic influence of the positively charged residues in S4, and the unexpectedly strong forces and effects on downstream TMHs elicited by amphipathic and re-entrant helices.

Biological membranes consist of a lipid bilayer studded with integral and peripheral membrane proteins. Most α-helical membrane proteins require protein-conducting insertases known as translocons to assist in their membrane insertion and folding. While the sequence-dependent propensities for a helix to either translocate through the translocon or insert into the membrane have been codified into numerical hydrophobicity scales, the corresponding propensity to partition into the membrane interface remains unrevealed. By engineering diagnostic glycosylation sites around test peptide sequences inserted into a host protein, we devised a system that can differentiate between water-soluble, surface-bound, and transmembrane (TM) states of the sequence based on its glycosylation pattern. Using this system, we determined the sequence-dependent propensities for transfer from the translocon to a TM, interfacial, or extramembrane space and compared these propensities with the corresponding probability distributions determined from the sequences and structures of experimentally determined proteins.

Secretory proteins are critically dependent on the correct processing of their signal sequence by the signal peptidase complex (SPC). This step, which is essential for the proper folding and localization of proteins in eukaryotic cells, is still not fully understood. In eukaryotes, the SPC comprises four evolutionarily conserved membrane subunits (Spc1-3 and Sec11). Here, we investigated the role of Spc2, examining SPC cleavage efficiency on various models and natural signal sequences in yeast cells depleted of or with mutations in Spc2. Our data show that discrimination between substrates and identification of the cleavage site by SPC is compromised when Spc2 is absent or mutated. Molecular dynamics simulation of the yeast SPC AlphaFold2-Multimer model indicates that membrane thinning at the center of SPC is reduced without Spc2, suggesting a molecular explanation for the altered substrate recognition properties of SPC lacking Spc2. These results provide new insights into the molecular mechanisms by which SPC governs protein biogenesis.

Nascent polypeptide chains (NCs) are extruded from the ribosome through an exit tunnel (ET) traversing the large ribosomal subunit. The ET's irregular and chemically complex wall allows for various NC-ET interactions. Translational arrest peptides (APs) bind in the ET to induce translational arrest, a property that can be exploited to study NC-ET interactions by Force Profile Analysis (FPA). We employed FPA and molecular dynamics (MD) simulations to investigate how individual residues placed in a glycine-serine repeat segment within an AP-stalled NC interact with the ET to exert a pulling force on the AP and release stalling. Our results indicate that large and hydrophobic residues generate a pulling force on the NC when placed ≳10 residues away from the peptidyl transfer center (PTC). Moreover, an asparagine placed 12 residues from the PTC makes a specific stabilizing interaction with the tip of ribosomal protein uL22 that reduces the pulling force on the NC, while a lysine or leucine residue in the same position increases the pulling force. Finally, the MD simulations suggest how the Mannheimia succiniproducens SecM AP interacts with the ET to promote translational stalling.

SignalP ( https://services.healthtech.dtu.dk/services/SignalP-6.0/ ) is a very popular prediction method for signal peptides, the intrinsic signals that make proteins secretory. The SignalP web server has existed since 1995 and is now in its sixth major version. In this historical account, we (three authors who have taken part in the entire journey plus the first author of the latest version) describe the differences between the versions and discuss the various decisions taken along the way.

In vivo, the majority of nascent protein chains must begin folding during translation in order to obtain their native structure. While the importance of co-translational folding has become increasingly clear, the specific mechanisms underlying the coordination between the ribosome, nascent chain and molecular chaperones are still uncertain. Here, we construct a model of the co-translational folding pathway of the calcium responsive domain (CRD) of the KV7.2 human neuronal ion channel, and demonstrate that calmodulin (CaM) is crucial. By combining Force Profile Analysis and single-molecule force spectroscopy techniques, we find that CaM, in a calcium-dependent manner, affects early folding events involving three key alpha-helices in the CRD. In addition, this study suggest that CaM at early stages induces the formation of metastable hairpins, as a part of the co-translational folding pathway. These findings expand on the role of CaM as a key regulator of folding in eukaryotes: not only as an essential cellular signaling protein, but also as a bona fide molecular chaperone. ### Competing Interest Statement The authors have declared no competing interest.

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.

Traditionally, protein folding has been analyzed through in vitro unfolding and refolding approaches, which fall short of emulating the co-translational folding events that occur within cellular environments. Consequently, our understanding of co-translational folding, particularly in ion channels and other proteins, remains limited. Our research has illuminated the pivotal role of calmodulin (CaM) in orchestrating the vectorial folding process of the calcium-responsive domain (CRD) within the Kv7.2 channel. We employ force profile analysis (FPA) to gauge the force applied to the nascent chain during the initial folding stages. In this study, we delineate the force profile of the CRD during translation, revealing that CaM is indispensable for instigating early folding events in the three pivotal helices: the IQ site of helix A, helix TW, and helix B. In a surprising turn, we observe that CaM also exerts influence on the non-target helices C and D, which constitute the subunit interaction domain of this channel. Furthermore, we have discovered that CaM assists in facilitating the co-translational folding process of the SK4 channel. This investigation sheds fresh light on the intricate role played by CaM in facilitating the co-translational folding mechanism of the Kv7.2 channel's C-terminal region and suggests potential involvement of CaM and calcium in the co-translational folding of other select proteins.

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.

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 E 14 in transmembrane helix 1 (TMH1) and S 64 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.

Signal peptides (SPs) are short amino acid sequences that control protein secretion and translocation in all living organisms. SPs can be predicted from sequence data, but existing algorithms are unable to detect all known types of SPs. We introduce SignalP 6.0, a machine learning model that detects all five SP types and is applicable to metagenomic data.

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.

Thanks to the rapid progress in membrane protein crystallography and cryogenic electron microscopy in recent years, we have become increasingly accustomed to seeing protein structures characterized by long transmembrane α-helices (TMHs) crisscrossing a lipid bilayer (1). But how is it decided which segments of the polypeptide form the TMHs, and what are the precise sequence requirements for their membrane insertion? The great majority of integral membrane proteins require one or another type of translocon to catalyze insertion into the target membrane. Themost commonly used and best understood of these are the universally conserved Sec-type translocons found in the cytoplasmic membrane of bacteria (the SecYEG complex) and the endoplasmic reticulum (ER) membrane in eukaryotes (the Sec61 complex) (2). They mediate cotranslational membrane insertion of the growing polypeptide as it emerges from the exit tunnel of translocon-bound ribosomes (3), and have an evolutionary conserved structure with a central channel through which hydrophilic polypeptide segments can be translocated across the membrane and a dynamic “lateral gate” through which hydrophobic segments in the nascent polypeptide chain can partition into the surrounding lipid bilayer to form TMHs (4, 5). Over the past two decades, statistical and experimental studies have progressively uncovered the sequence characteristics that define the difference between polypeptide segments that can form TMHs and those that cannot. To a first approximation, the efficiency of membrane insertion of a TMH is dictated by its hydrophobicity, although interactions with neighboring TMHs, or sequence variations in segments immediately flanking the TMH, can shift the hydrophobicity threshold (HT) required for membrane insertion (6). In PNAS, Janoschke et al. (7) now uncover another characteristic of the nascent polypeptide chain that can affect the HT: the folding properties of the region immediately upstream of the TMH. In their study, Janoschke et al. (7) take, as their point of departure, an observation made in 1996 by Kuroiwa et al. (8), who found that the introduction of progressively longer, presumably not well-folded, segments A