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 K_V7.2 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 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.

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.

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.

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.

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. 

The central themes of Cell Boundaries concern the structural and organizational principles underlying cell membranes, and how these principles enable function. By building a biological and biophysical foundation for understanding the organization of lipids in bilayers and the folding, assembly, stability, and function of membrane proteins, the book aims to broaden the knowledge of bioscience students to include the basic physics and physical chemistry that inform us about membranes. In doing so, it is hoped that physics students will find familiar territory that will lead them to an interest in biology. Our progress toward understanding membranes and membrane proteins depends strongly upon the concerted use of both biology and physics. It is important for students to know not only what we know, but how we have come to know it, so Cell Boundaries endeavours to bring out the history behind the central discoveries, especially in the early chapters, where the foundation is laid for later chapters. Science is far more interesting if, as students, we can appreciate and share in the adventures-and misadventures-of discovering new scientific knowledge. Cell Boundaries was written with advanced undergraduates and beginning graduate students in the biological and physical sciences in mind, though this textbook will likely have appeal to researchers and other academics as well. Highlights the history of important central discoveries Early chapters lay the foundation for later chapters to build on, so knowledge is amassed High-quality line diagrams illustrate key concepts and illuminate molecular mechanisms Box features and spreads expand on topics in main text, including histories of discoveries, special techniques, and applications. 

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. 

The ribosome stalling mechanism is a crucial biological process, yet its atomistic underpinning is still elusive. In this framework, the human XBP1u translational arrest peptide (AP) plays a central role in regulating the unfolded protein response (UPR) in eukaryotic cells. Here, we report multimicrosecond all-atom molecular dynamics simulations designed to probe the interactions between the XBP1u AP and the mammalian ribosome exit tunnel, both for the wild type AP and for four mutant variants of different arrest potencies. Enhanced sampling simulations allow investigating the AP release process of the different variants, shedding light on this complex mechanism. The present outcomes are in qualitative/quantitative agreement with available experimental data. In conclusion, we provide an unprecedented atomistic picture of this biological process and clear-cut insights into the key AP-ribosome interactions.