Gunnar von Heijne

Most proteins start to fold cotranslationally as they come off the ribosome. So far, studies of cotranslational folding have focused mainly on small, single-domain proteins. Here, we have used Force Profile Analysis to study the cotranslational folding of Firefly Luciferase, a complex 550-residue protein composed of an N-terminal domain (NTD) encompassing two split Rossmann folds (RF-1, RF-2) and a β-roll, and a flexibly attached C-terminal domain (CTD). The folding process is characterized by a quasi-continuous series of compaction/folding steps that generate intermediate-size pulling forces on the nascent chain, punctuated by a prominent high-force event that represents the folding of the RF-2 domain, and a few low-force instances that likely indicate the formation of distinct folding intermediates. Trigger Factor interacts extensively with the nascent chain when the central part of RF-2 and the early parts of the CTD are synthesized. Our analysis uncovers a cotranslational compaction/folding process that is rich in detail and not just a simple succession of a few distinct, cooperative folding transitions.

Protein secretion is essential for cell function and begins when signal peptides direct nascent proteins into the secretory pathway, where they are cleaved by the signal peptidase complex (SPC). Although this pathway is highly conserved, signal peptides do not always function efficiently across species, and the molecular basis for this incompatibility remains unclear. Here, we show that human signal peptides with longer hydrophobic cores efficiently target and translocate nascent proteins in both yeast and human but are frequently left uncleaved in yeast, identifying signal peptide cleavage as a major barrier to cross-species compatibility. Molecular dynamics (MD) simulations reveal that, despite their highly conserved architectures, yeast and human SPCs generate distinct membrane-thinning profiles near the signal peptide-binding region. Our results support a model in which SPC–lipid interactions tune the local membrane environment to accommodate signal peptides of distinct hydrophobic core length, providing a biophysical mechanism for species-specific signal peptide recognition. These findings offer a mechanistic framework for understanding and potentially engineering protein secretion across diverse expression hosts.

Most proteins start to fold cotranslationally as they come off the ribosome. So far, studies of cotranslational folding have focused mainly on small, single-domain proteins. Here, we have used Force Profile Analysis to study the cotranslational folding of Firefly luciferase, a complex 550-residue protein composed of an N-terminal domain (NTD) encompassing two split Rossmann folds (RF-1, RF-2) and a β-roll, and a flexibly attached C-terminal domain (CTD). The folding process is characterized by a quasi-continuous series of compaction/folding steps that generate intermediate-size pulling forces on the nascent chain, punctuated by a prominent high-force event that represents the folding of the RF-2 domain, and a few low-force instances that likely indicate the formation of distinct folding intermediates. Trigger Factor interacts extensively with the nascent chain when the central part of RF-2 and the early parts of the CTD are synthesized. Our analysis uncovers a cotranslational compaction/folding process that is rich in detail and not just a simple succession of a few distinct, cooperative folding transitions.

Viral protein biogenesis underpins every viral life cycle stage, and elucidating these processes could reveal fundamental principles of virus–host interaction, and vulnerabilities amenable to therapeutic targeting. Here we apply biophysical, molecular, and cell biology techniques to investigate the insertion, folding, and oligomerization of the SARS-CoV-2 M protein. We describe the sequential co-translational insertion of the hydrophobic core, and demonstrate that the cytosolic C-terminal domain undergoes slower adoption of its tertiary structure. Additionally, we characterize how the transmembrane domain bundle facilitates M-protein oligomerization. Our results reveal a hydrophobic residue cluster that is essential for protein folding and co-translational dimerization. Additionally, we identify the cellular machinery responsible for targeting and inserting the M protein into the ER membrane, and chaperones and cofactors that may contribute to proper 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.

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 unraveled. 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. UMAP analysis of a large collection of TM and water-soluble helices provide useful embeddings for analysis of these propensities and aid in understanding the physical properties and functions of antimicrobial, lytic, and fusogenic peptides.