Biology subjects

von Heijne, G.

Publications and source records attributed to von Heijne, G..

16 recordsLinked to original sources

bioRxiv2026

Buried Ion-Pairs Reshape Folding Pathways of Artificial Proteins

Ion-pairs are central charged motifs that control the biological function of several proteins. Yet, the physical principles linked to the formation of ion-pairs during protein folding remain poorly understood. Here, we show that buried ion-pairs of de novo-designed helical bundle proteins reshape the folding pathways by introducing kinetic barriers that are overcome by a hydrophobic collapse linked to packing of the protein core. By combining protein design with co-translational folding studies, nuclear magnetic resonance (NMR) spectroscopy, and molecular dynamics simulations, we show that introduction of a buried ion-pair leads to a shifted folding transition that balances desolvation and electrostatic interactions within the protein and the bulk solvent. Moreover, we find that the folding pathway is sensitive to modifications in non-polar interactions and motifs that electrostatically shield the ion-pair from its surroundings. Our combined findings provide a blueprint for understanding how functional motifs affect protein folding and how mutation of charged networks alter the stability of proteins.

biophysics↗

bioRxiv2026

Quasi-continuous cotranslational compaction and folding of a multidomain protein

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 {beta}-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.

molecular biology↗

bioRxiv2026

SARS-CoV-2 membrane protein biogenesis

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.

molecular biology↗

bioRxiv2025

Cotranslational folding and maturation of HIV-1 protease

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 -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.

molecular biology↗

bioRxiv2024

Calmodulin acts as a chaperone during co-translational folding of the Kv7.2 channel Calcium Responsive Domain.

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 have constructed a model of the co-translational folding pathway of the calcium responsive domain (CRD) of the human neuronal KV7.2 human neuronal ion channel, showing that calmodulin (CaM) is crucial. 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 -helices in the CRD. In addition, this study suggests 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 co-translational molecular chaperone.

molecular biology↗

bioRxiv2024

Cotranslational membrane insertion of the voltage-sensitive K+ channel KvAP

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 to follow the cotranslational insertion of the six-TMH KvAP voltage-sensitive ion channel into the E. 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.

molecular biology↗

bioRxiv2022

Cotranslational folding of human growth hormone in vitro and in Escherichia coli

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 E. 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.

molecular biology↗

bioRxiv2022

Cotranslational folding and assembly of the dimeric E. coli inner membrane protein EmrE

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 possible also for integral membrane proteins is unknown, however. 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 E. 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 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 can thus both start to fold and start to dimerize during the cotranslational membrane-insertion process. Significance statementMany water-soluble proteins are known to fold and even dimerize cotranslationally, i.e., when still attached to the ribosome. Here, we show that an E. coli inner membrane protein can also start to fold and dimerize cotranslationally, establishing the generality of these cotranslational maturation processes.

biochemistry↗

bioRxiv2021

Upstream charged and hydrophobic residues impact the timing of membrane insertion of transmembrane helices

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 center. 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 BtuC protein: 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.

biochemistry↗

bioRxiv2021

Probing interplays between human XBP1u translational arrest peptide and 80S ribosome

The ribosome stalling mechanism is a crucial biological process; yet its atomistic underpinning is still elusive. In this framework, the XBP1u translational arrest peptide (AP) plays a central role in regulating the Unfolded Protein Response (UPR) in eukaryotic cells. Here, we report multi-microseconds 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 potency. 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.

molecular biology↗

bioRxiv2020

Cotranslational folding of alkaline phosphatase in the periplasm of Escherichia coli

Cotranslational protein folding studies using Force Profile Analysis, a method where the SecM translational arrest peptide is used to detect folding-induced forces acting on the nascent polypeptide, have so far been limited mainly to small domains of cytosolic proteins that fold in close proximity to the translating ribosome. In this study, we investigate the cotranslational folding of the periplasmic, disulfide bond-containing E. coli protein alkaline phosphatase (PhoA) in a wild-type strain background and a strain background devoid of the periplasmic thiol:disulfide interchange protein DsbA. We find that folding-induced forces can be transmitted via the nascent chain from the periplasm to the polypeptide transferase center in the ribosome, a distance of ~160 Å, and that PhoA appears to fold cotranslationally via at least two disulfide-stabilized folding intermediates. Thus, Force Profile Analysis can be used to study cotranslational folding of proteins in an extra-cytosolic compartment, like the periplasm.Competing Interest StatementThe authors have declared no competing interest.View Full Text

molecular biology↗

bioRxiv2020

The ribosome modulates folding inside the ribosomal exit tunnel

Proteins commonly fold cotranslationally on the ribosome, while the nascent chain emerges from the ribosomal tunnel. Protein domains that are sufficiently small can even fold while still located inside the tunnel. However, the effect of the tunnel on the folding dynamics of these domains is still not well understood. Here, we combine optical tweezers with single-molecule FRET and molecular dynamics simulations to investigate folding of the small zinc-finger domain ADR1a inside and at the vestibule of the ribosomal tunnel. The tunnel is found to accelerate folding and stabilize the folded state, reminiscent of the effects of chaperonins. However, a simple mechanism involving stabilization by confinement does not reproduce the results. Instead, it appears that electrostatic interactions between the protein and ribosome contribute to the observed folding acceleration and stabilization of ADR1a.Competing Interest StatementThe authors have declared no competing interest.View Full Text

biophysics↗

bioRxiv2019

Detecting Novel Sequence Signals in Targeting Peptides Using Deep Learning

In bioinformatics, machine learning methods have been used to predict features embedded in the sequences. In contrast to what is generally assumed, machine learning approaches can also provide new insights into the underlying biology. Here, we demonstrate this by presenting TargetP 2.0, a novel state of art method to identify N-terminal sorting signals, which direct proteins to the secretory pathway, mitochondria and chloroplasts or other plastids.\n\nBy examining the strongest signals from the attention layer in the network, we find that the second residue in the protein, i.e. the one following the initial methionine, has a strong influence on the classification. When subsequently examining all targeting peptides, we observe that two-thirds of chloroplast and thylakoid transit peptides have an alanine in position two, but only 20% of other plant proteins. Further highlighting the importance of the second residue, we also note that in fungi and single-celled eukaryotes, less than 30% of the targeting peptides have an amino acid that allows the removal of the N-terminal methionine compared with 60% for the proteins without targeting peptide.\n\nTargetP 2.0 is available at http://www.cbs.dtu.dk/services/TargetP-2.0/index.php

bioinformatics↗

bioRxiv2018

Force-profile analysis of the cotranslational folding of HemK and filamin domains: Comparison of biochemical and biophysical folding assays

We have characterized the cotranslational folding of two small protein domains of different folds - the a-helical N-terminal domain of HemK and the {beta}-rich FLN5 filamin domain - by measuring the force that the folding protein exerts on the nascent chain when located in different parts of the ribosome exit tunnel (Force-Profile Analysis - FPA), allowing us to compare FPA to three other techniques currently used to study cotranslational folding: real-time FRET, PET, and NMR. We find that FPA identifies the same cotranslational folding transitions as do the other methods, and that these techniques therefore reflect the same basic process of cotranslational folding in similar ways.

molecular biology↗

bioRxiv2018

Forces on nascent polypeptides during membrane insertion and translocation via the Sec translocon

During ribosomal translation, nascent polypeptide chains (NCs) undergo a variety of physical processes that determine their fate in the cell. Translation arrest peptide (AP) experiments are used to measure the external pulling forces that are exerted on the NC at different lengths during translation. To elucidate the molecular origins of these forces, a recently developed coarsegrained molecular dynamics (CGMD) is used to directly simulate the observed pulling-force profiles, thereby disentangling contributions from NC-translocon and NC-ribosome interactions, membrane partitioning, and electrostatic coupling to the membrane potential. This combination of experiment and theory reveals mechanistic features of Sec-facilitated membrane integration and protein translocation, including the interplay between transient interactions and conformational changes that occur during ribosomal translation to govern protein biogenesis.

biophysics↗

bioRxiv2017

Stable Membrane Topologies Of Small Dual-Topology Membrane Proteins

The topologies of -helical membrane proteins are generally thought to be determined during their cotranslational insertion into the membrane. It is typically assumed that membrane topologies remain static after this process has ended. Recent findings, however, question this static view by suggesting that some parts of, or even the whole protein, can reorient in the membrane on a biologically relevant time scale. Here, we focus on anti-parallel homo-or hetero-dimeric Small Multidrug Resistance proteins, and examine whether the individual monomers can undergo reversible topological inversion (flip-flop) in the membrane until they are trapped in a fixed orientation by dimerization. By perturbing dimerization using various means, we show that the membrane topology of a monomer is unaffected by the presence or absence of its dimerization partner. Thus, membrane-inserted monomers attain their final topologies independently of dimerization, suggesting that wholesale topological inversion is an unlikely event in vivo.

biochemistry↗