Proteins that fold cotranslationally may do so in a restricted configurational space, due to the volume occupied by ribosome. How does this environment, coupled with close proximity of ribosome, affect folding pathway protein? Previous studies have shown cotranslational process for many proteins, including small, single domains, is directly affected Here, we investigate an all-β Ig domain, titin I27. Using arrest peptide-based assay and structural cryo-EM, show I27 folds mouth ribosome exit...

Protein insertion into the bacterial inner membrane is facilitated by SecYEG or YidC. Although most likely constitutes major integration site, small proteins have been shown to integrate via We show that YidC can also multispanning such as mannitol permease TatC, which had considered be exclusively integrated SecYEG. Only SecA-dependent strictly require for integration, suggests SecA only interact with translocon, but not insertase. Targeting of mediated signal recognition particle (SRP),...

The E. coli ribosome exit tunnel can accommodate small folded proteins, while larger ones fold outside. It remains unclear, however, to what extent the geometry of influences protein folding. Here, using ribosomes with deletions in loops proteins uL23 and uL24 that protrude into tunnel, we investigate how determines where different sizes fold. We find a 29-residue zinc-finger domain normally folding close loop folds deeper Δloop ribosomes, two ~ 100 residue near port at locations good...

Abstract Proteins commonly fold co-translationally at the ribosome, while nascent chain emerges from ribosomal exit tunnel. Protein domains that are sufficiently small can even still located inside However, effect of tunnel on folding dynamics these is not well understood. Here, we combine optical tweezers with single-molecule FRET and molecular simulations to investigate zinc-finger domain ADR1a vestibule The found accelerate stabilize folded state, reminiscent effects chaperonins. a simple...

Protein secretion in Gram-negative bacteria is essential for both cell viability and pathogenesis. The vast majority of secreted proteins exit the cytoplasm through a transmembrane conduit called Sec translocon process that facilitated by ancillary modules, such as SecA, SecDF-YajC, YidC, PpiD. In this study we have characterized YfgM, protein with no annotated function. We found it to be novel subunit co-purifies PpiD SecYEG after immunoprecipitation blue native/SDS-PAGE. Phenotypic...

The Sec translocon constitutes a ubiquitous protein transport channel that consists in bacteria of the three core components: SecY, SecE, and SecG. Additional proteins interact with SecYEG during different stages transport. During targeting, interacts SecA, SRP receptor, or ribosome. Protein into across membrane is then facilitated by interaction YidC SecDFYajC complex. transport, likely to also quality control machinery, but details about this are missing. By vivo vitro site-directed...

Abstract In Escherichia coli, elevated levels of free l-tryptophan (l-Trp) promote translational arrest the TnaC peptide by inhibiting its termination. However, mechanism which translation-termination UGA-specific decoding release factor 2 (RF2) is inhibited at UGA stop codon stalled TnaC-ribosome-nascent chain complexes has so far been ambiguous. This study presents cryo-EM structures for ribosomes in absence and presence RF2 average resolutions 2.9 3.5 Å, respectively. Stalled assumes a...

Abstract 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 nascent polypeptide, have so far been limited mainly small domains of cytosolic proteins that fold in close proximity translating ribosome. In this study, we investigate cotranslational periplasmic, disulfide bond‐containing Escherichia coli alkaline phosphatase (PhoA) wild‐type strain background and devoid...

In Gram-negative bacteria, periplasmic domains in inner membrane proteins are cotranslationally translocated across the through SecYEG translocon. To what degree such also start to fold is generally difficult determine using currently available methods. Here, we apply Force Profile Analysis (FPA) - a method where translational arrest peptide used detect folding-induced forces acting on nascent polypeptide follow cotranslational translocation and folding of large domain E. coli protease LepB...

VOLUME 288 (2013) PAGES 16295–16307 PAGE 16301: The immunoblot data in Fig. 4A was not obtained from a conditional secDF depletion strain Escherichia coli BL325 with plasmid-borne copy of SecY(I91pBpa)EG as stated. correct images representing SecD, SecF, SecY, and YidC levels inner membrane vesicles the E. are now shown. This correction does affect interpretation results or conclusions this work.

The malarial parasite Plasmodium exports its own proteins to the cell surfaces of red blood cells (RBCs) during infection. Examples exported include members repetitive interspersed family (RIFIN) and subtelomeric variable open reading frame (STEVOR) from falciparum. presence these parasite-derived on infected RBCs triggers adhesion uninfected (rosetting) vascular endothelium potentially obstructing flow. While there is a fair amount information localization RBCs, less known about how they...

Abstract Proteins that fold cotranslationally may do so in a restricted configurational space, due to the volume occupied by ribosome. How does this environment, coupled with close proximity of ribosome, affect folding pathway protein? Previous studies have shown cotranslational process for many proteins, including small, single domains, is directly affected Here, we investigate an all-b immunoglobulin domain, titin I27. Using arrest peptide-based assay and structural cryo-EM, show I27 folds...

Abstract The E.coli ribosome exit tunnel can accommodate small folded proteins, while larger ones fold outside. It remains unclear, however, to what extent the geometry of influences protein folding. Here, using E. coli ribosomes with deletions in loops proteins uL23 and uL24 that protrude into tunnel, we investigate how determines where different sizes fold. We find a 29-residue zinc-finger domain normally folding close loop folds deeper Δloop ribosomes, two ~100-residue near port at...

Proteins commonly fold cotranslationally on the ribosome, while nascent chain emerges from ribosomal tunnel. Protein domains that are sufficiently small can even still located inside However, effect of tunnel folding dynamics these is not well understood. Here, we combine optical tweezers with single-molecule FRET and molecular simulations to investigate zinc-finger domain ADR1a at vestibule The found accelerate stabilize folded state, reminiscent effects chaperonins. a simple mechanism...

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

Abstract Nascent polypeptide chains (NCs) are extruded from the ribosome through an exit tunnel (ET) traversing large ribosomal subunit. The ET’s irregular and chemically complex wall allows for various NC-ET interactions. Translational arrest peptides (APs) bind in ET to induce translational arrest, a property that can be exploited study interactions by Force Profile Analysis (FPA). We employed FPA molecular dynamics (MD) simulations investigate how individual residues placed glycine-serine...

ABSTRACT The malarial parasite Plasmodium , infects red blood cells by remodeling them and transporting its own proteins to their cell surface. These trigger adhesion of infected uninfected (rosetting), the vascular endothelium, obstructing flow contributing pathogenesis. RIFINs ( P. falciparum -encoded repetitive interspersed families polypeptides) STEVORs (subtelomeric variable open reading frame), are two classes that involved in rosetting. Here we study membrane insertion topology three...

This protocol describes the procedure make an S30 derived E.coli cell lysate that lacks membrane components. Modified from a published in https://doi.org/10.1091/mbc.e11-07-0590 The takes three days total, including preparation time.

Isolation of ribosome-nascent chains co-translationally folded domains. Detailed description protocol published in https://doi.org/10.1073/pnas.1810523115. The arrest sequence used for stalling was TnaC (the leader peptide the tryptophanase operon).