Publications

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.

Several laboratories have carried out molecular dynamics (MD) simulations of arginine interactions with lipid bilayers and found that the energetic cost of placing arginine in lipid bilayers is an order of magnitude greater than observed in molecular biology experiments in which Arg-containing transmembrane helices are inserted across the endoplasmic reticulum membrane by the Sec61 translocon. We attempt here to reconcile the results of the two approaches. We first present MD simulations of guanidinium groups alone in lipid bilayers, and then, to mimic the molecular biology experiments, we present simulations of hydrophobic helices containing single Arg residues at different positions along the helix. We discuss the simulation results in the context of molecular biology results and show that the energetic discrepancy is reduced, but not eliminated, by considering free energy differences between Arg at the interface and at the center of the model helices. The reduction occurs because Arg snorkeling to the interface prevents Arg from residing in the bilayer center where the energetic cost of desolvation is highest. We then show that the problem with MD simulations is that they measure water-to-bilayer free energies, whereas the molecular biology experiments measure the energetics of partitioning from translocon to bilayer, which raises the fundamental question of the relationship between water-to-bilayer and water-to-translocon partitioning. We present two thermodynamic scenarios as a foundation for reconciliation of the simulation and molecular biology results. The simplest scenario is that translocon-to-bilayer partitioning is independent of water-to-bilayer partitioning; there is no thermodynamic cycle connecting the two paths.

It is becoming increasingly clear that many proteins start to fold cotranslationally, before the entire polypeptide chain has been synthesized on the ribosome. One class of proteins that a priori would seem particularly prone to cotranslational folding is repeat proteins, i.e ., proteins that are built from an array of nearly identical sequence repeats. However, while the folding of repeat proteins has been studied extensively in vitro with purified proteins, only a handful of studies have addressed the issue of cotranslational folding of repeat proteins. Here, we have determined the structure and studied the cotranslational folding of a β-helix pentarepeat protein from the human pathogen Clostridium botulinum – a homolog of the Fluoroquinolone Resistance Protein MfpA – using an assay in which the SecM translational arrest peptide serves as a force sensor to detect folding events. We find that cotranslational folding of a segment corresponding to the first four of the eight β-helix coils in the protein produces enough force to release ribosome stalling, and that folding starts when this unit is ~35 residues away from the P-site, near the distal end of the ribosome exit tunnel. An additional folding transition is seen when the whole PENT moiety emerges from the exit tunnel. The early cotranslational formation of a folded unit may be important to avoid misfolding events in vivo , and may reflect the minimal size of a stable β-helix since it is structurally homologous to the smallest known β-helix protein, a four-coil protein that is stable in solution.

We have studied the effects of single charged residues on the position of a model transmembrane helix in the endoplasmic reticulum membrane using the glycosylation mapping technique. Asp and Glu residues cause a re-positioning of the C-terminal end of the transmembrane helix when placed in the one to two C-terminal turns but not when placed more centrally. Arg and Lys residues, in contrast, have little effect when placed in the two C-terminal turn but give rise to a more substantial shift in position when placed 9–11 residues from the helix end. We suggest that this difference between the effects of positively and negatively charged residues can be explained by the so-called snorkel effect, i.e. that the very long side-chains of Arg and Lys can reach up along the transmembrane helix to allow the terminal, charged moiety to reside in the lipid headgroup region while the Cα of the residue is positioned well below the membrane/water interface.

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.

We have used the natural N‐glycosylation site in the N‐tail of cig30, a eukaryotic polytopic membrane protein, as a marker for N‐tail translocation across the microsomal membrane. Analysis of C‐terminally truncated cig30 constructs reveals that the first transmembrane segment is sufficient for translocation of the wild‐type N‐tail; in contrast, in a mutant with four arginines introduced into the N‐tail the second transmembrane segment is also required for efficient N‐tail translocation. Our observations imply a non‐sequential assembly mechanism in which the ultimate location of the N‐tail relative to the membrane may depend on more than one transmembrane segment.

No abstract is provided for this article.

Background: EmrE is a dual-topology membrane protein, but it is not clear whether the active form is composed of parallel or anti-parallel dimers. Results: Antiparallel EmrE dimers are more stable than parallel dimers. Conclusion: Antiparallel EmrE dimers most likely correspond to the functional form of the protein. Significance: The results provide new information on the functional form of EmrE.