Cotranslational folding of small protein domains within the ribosome exit tunnel may be an important cellular strategy to avoid protein misfolding. However, the pathway of cotranslational folding has ...so far been described only for a few proteins, and therefore, it is unclear whether folding in the ribosome exit tunnel is a common feature for small protein domains. Here, we have analyzed nine small protein domains and determined at which point during translation their folding generates sufficient force on the nascent chain to release translational arrest by the SecM arrest peptide, both in vitro and in live E. coli cells. We find that all nine protein domains initiate folding while still located well within the ribosome exit tunnel.
Signal peptides (SPs) are short amino acid sequences in the amino terminus of many newly synthesized proteins that target proteins into, or across, membranes. Bioinformatic tools can predict SPs from ...amino acid sequences, but most cannot distinguish between various types of signal peptides. We present a deep neural network-based approach that improves SP prediction across all domains of life and distinguishes between three types of prokaryotic SPs.
Membrane-protein topology von Heijne, Gunnar
Nature reviews. Molecular cell biology,
12/2006, Letnik:
7, Številka:
12
Journal Article
Recenzirano
In the world of membrane proteins, topology defines an important halfway house between the amino-acid sequence and the fully folded three-dimensional structure. Although the concept of ...membrane-protein topology dates back at least 30 years, recent advances in the field of translocon-mediated membrane-protein assembly, proteome-wide studies of membrane-protein topology and an exponentially growing number of high-resolution membrane-protein structures have given us a deeper understanding of how topology is determined and of how it evolves.
Since high-resolution structural data are still scarce, different kinds of theoretical structure prediction algorithms are of major importance in membrane protein biochemistry. But how well do the ...current prediction methods perform? Which structural features can be predicted and which cannot? And what can we expect in the next few years?
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.
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 show that hGH starts to fold before it is completely released from the ribosome, and that it can interact with chaperones during folding.
At what point during translation do proteins fold? It is well established that proteins can fold cotranslationally outside the ribosome exit tunnel, whereas studies of folding inside the exit tunnel ...have so far detected only the formation of helical secondary structure and collapsed or partially structured folding intermediates. Here, using a combination of cotranslational nascent chain force measurements, inter-subunit fluorescence resonance energy transfer studies on single translating ribosomes, molecular dynamics simulations, and cryoelectron microscopy, we show that a small zinc-finger domain protein can fold deep inside the vestibule of the ribosome exit tunnel. Thus, for small protein domains, the ribosome itself can provide the kind of sheltered folding environment that chaperones provide for larger proteins.
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•Cotranslational folding is studied by arrest-peptide-mediated force measurements•Single-molecule measurements show that a pulling force prevents ribosome stalling•A ribosome-tethered zinc-finger domain is visualized by cryo-EM (electron microscopy)•The zinc-finger domain is shown to fold deep inside the ribosome exit tunnel
Nilsson et al. present an integrated approach to the study of cotranslational protein folding, in which the folding transition is mapped by arrest-peptide-mediated force measurements, molecular dynamics simulations, and cryo-EM (electron microscopy). The small zinc-finger domain ADR1a is shown to fold deep inside the ribosome exit tunnel.
The biogenesis, folding, and structure of α-helical membrane proteins (MPs) are important to understand because they underlie virtually all physiological processes in cells including key metabolic ...pathways, such as the respiratory chain and the photosystems, as well as the transport of solutes and signals across membranes. Nearly all MPs require translocons—often referred to as protein-conducting channels—for proper insertion into their target membrane. Remarkable progress toward understanding the structure and functioning of translocons has been made during the past decade. Here, we review and assess this progress critically. All available evidence indicates that MPs are equilibrium structures that achieve their final structural states by folding along thermodynamically controlled pathways. The main challenge for cells is the targeting and membrane insertion of highly hydrophobic amino acid sequences. Targeting and insertion are managed in cells principally by interactions between ribosomes and membrane-embedded translocons. Our review examines the biophysical and biological boundaries of MP insertion and the folding of polytopic MPs in vivo. A theme of the review is the under-appreciated role of basic thermodynamic principles in MP folding and assembly. Thermodynamics not only dictates the final folded structure but also is the driving force for the evolution of the ribosome–translocon system of assembly. We conclude the review with a perspective suggesting a new view of translocon-guided MP insertion.
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•Helical MPs are inserted co-translationally by ribosomes docked to translocons.•Recent ribosome–translocon structures provide dramatic insights into MP assembly.•Folding of inserted MPs is driven by strong thermodynamic forces.•A new view of translocon-guided MP folding is presented.
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.
During cotranslational membrane insertion, transmembrane helices first make contact with the membrane when their N‐terminal end is ~ 45 residues away from the peptidyl transferase centre. Here, we show that upstream positively charged and re‐entrant loops in the Escherichia coli inner membrane protein BtuC can delay the first contact. Thus, both electrostatic and hydrophobic interactions involving upstream sequence elements can impact membrane insertion.
Signal recognition particle (SRP) is a universally conserved protein-RNA complex that mediates co-translational protein translocation and membrane insertion by targeting translating ribosomes to ...membrane translocons. The existence of parallel co- and post-translational transport pathways, however, raises the question of the cellular substrate pool of SRP and the molecular basis of substrate selection. Here we determine the binding sites of bacterial SRP within the nascent proteome of Escherichia coli at amino acid resolution, by sequencing messenger RNA footprints of ribosome-nascent-chain complexes associated with SRP. SRP, on the basis of its strong preference for hydrophobic transmembrane domains (TMDs), constitutes a compartment-specific targeting factor for nascent inner membrane proteins (IMPs) that efficiently excludes signal-sequence-containing precursors of periplasmic and outer membrane proteins. SRP associates with hydrophobic TMDs enriched in consecutive stretches of hydrophobic and bulky aromatic amino acids immediately on their emergence from the ribosomal exit tunnel. By contrast with current models, N-terminal TMDs are frequently skipped and TMDs internal to the polypeptide sequence are selectively recognized. Furthermore, SRP binds several TMDs in many multi-spanning membrane proteins, suggesting cycles of SRP-mediated membrane targeting. SRP-mediated targeting is not accompanied by a transient slowdown of translation and is not influenced by the ribosome-associated chaperone trigger factor (TF), which has a distinct substrate pool and acts at different stages during translation. Overall, our proteome-wide data set of SRP-binding sites reveals the underlying principles of pathway decisions for nascent chains in bacteria, with SRP acting as the dominant triaging factor, sufficient to separate IMPs from substrates of the SecA-SecB post-translational translocation and TF-assisted cytosolic protein folding pathways.
Abstract
In Escherichia coli, elevated levels of free l-tryptophan (l-Trp) promote translational arrest of the TnaC peptide by inhibiting its termination. However, the mechanism by which ...translation-termination by the UGA-specific decoding release factor 2 (RF2) is inhibited at the UGA stop codon of stalled TnaC-ribosome-nascent chain complexes has so far been ambiguous. This study presents cryo-EM structures for ribosomes stalled by TnaC in the absence and presence of RF2 at average resolutions of 2.9 and 3.5 Å, respectively. Stalled TnaC assumes a distinct conformation composed of two small α-helices that act together with residues in the peptide exit tunnel (PET) to coordinate a single L-Trp molecule. In addition, while the peptidyl-transferase center (PTC) is locked in a conformation that allows RF2 to adopt its canonical position in the ribosome, it prevents the conserved and catalytically essential GGQ motif of RF2 from adopting its active conformation in the PTC. This explains how translation of the TnaC peptide effectively allows the ribosome to function as a L-Trp-specific small-molecule sensor that regulates the tnaCAB operon.
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