| 1. |
- Cymer, Florian, et al.
(författare)
-
Exploration of the Arrest Peptide Sequence Space Reveals Arrest-enhanced Variants
- 2015
-
Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 290:16, s. 10208-10215
-
Tidskriftsartikel (refereegranskat)abstract
- Translational arrest peptides (APs) are short stretches of polypeptides that induce translational stalling when synthesized on a ribosome. Mechanical pulling forces acting on the nascent chain can weaken or even abolish stalling. APs can therefore be used as in vivo force sensors, making it possible to measure the forces that act on a nascent chain during translation with single-residue resolution. It is also possible to score the relative strengths of APs by subjecting them to a given pulling force and ranking them according to stalling efficiency. Using the latter approach, we now report an extensive mutagenesis scan of a strong mutant variant of the Mannheimia succiniciproducens SecM AP and identify mutations that further increase the stalling efficiency. Combining three such mutations, we designed an AP that withstands the strongest pulling force we are able to generate at present. We further show that diproline stretches in a nascent protein act as very strong APs when translation is carried out in the absence of elongation factor P. Our findings highlight critical residues in APs, show that certain amino acid sequences induce very strong translational arrest and provide a toolbox of APs of varying strengths that can be used for in vivo force measurements.
|
|
| 2. |
- Cymer, Florian, et al.
(författare)
-
Weak pulling forces exerted on N-in-orientated transmembrane segments during co-translational insertion into the inner membrane of Escherichia coli
- 2014
-
Ingår i: FEBS Letters. - : Wiley. - 0014-5793 .- 1873-3468. ; 588:10, s. 1930-1934
-
Tidskriftsartikel (refereegranskat)abstract
- Transmembrane helices (TMHs) in membrane proteins can be orientated with their N-terminus towards the cytoplasm (N-in), or facing the non-cytoplasmic side (N-out). Most membrane proteins are inserted co-translationally into membranes, aided by Sec-type translocons. Since the final orientation of N-in-and N-out-orientated TMHs differs, they could also interact differently with the translocon and the surrounding membrane during insertion. We measured pulling forces exerted on N-in-orientated TMHs during co-translational insertion into the inner membrane of Escherichia coli. Our results demonstrate that Nin-orientated TMHs experience a weaker pulling force but retain the overall biphasic force profile seen previously for Nout-orientated TMHs (Ismail et al., 2012 [1]).
|
|
| 3. |
- Ismail, Nurzian, et al.
(författare)
-
A biphasic pulling force acts on transmembrane helices during translocon mediated membrane integration
- 2012
-
Ingår i: Nature Structural & Molecular Biology. - : Springer Science and Business Media LLC. - 1545-9993 .- 1545-9985. ; 19:10, s. 1018-1022
-
Tidskriftsartikel (refereegranskat)abstract
- Membrane proteins destined for insertion into the inner membrane of bacteria or the endoplasmic reticulum membrane in eukaryotic cells are synthesized by ribosomes bound to the bacterial SecYEG or the homologous eukaryotic Sec61 translocon. During co-translational membrane integration, transmembrane alpha-helical segments in the nascent chain exit the translocon through a lateral gate that opens toward the surrounding membrane, but the mechanism of lateral exit is not well understood. In particular, little is known about how a transmembrane helix behaves when entering and exiting the translocon. Using translation-arrest peptides from bacterial SecM proteins and from the mammalian Xbp1 protein as force sensors, we show that substantial force is exerted on a transmembrane helix at two distinct points during its transit through the translocon channel, providing direct insight into the dynamics of membrane integration.
|
|
| 4. |
- Ismail, Nurzian, et al.
(författare)
-
Charge-driven dynamics of nascent-chain movement through the SecYEG translocon
- 2015
-
Ingår i: Nature Structural & Molecular Biology. - : Springer Science and Business Media LLC. - 1545-9993 .- 1545-9985. ; 22:2, s. 145-149
-
Tidskriftsartikel (refereegranskat)abstract
- On average, every fifth residue in secretory proteins carries either a positive or a negative charge. In a bacterium such as Escherichia coli, charged residues are exposed to an electric field as they transit through the inner membrane, and this should generate a fluctuating electric force on a translocating nascent chain. Here, we have used translational arrest peptides as in vivo force sensors to measure this electric force during cotranslational chain translocation through the SecYEG translocon. We find that charged residues experience a biphasic electric force as they move across the membrane, including an early component with a maximum when they are 47-49 residues away from the ribosomal P site, followed by a more slowly varying component. The early component is generated by the transmembrane electric potential, whereas the second may reflect interactions between charged residues and the periplasmic membrane surface.
|
|
| 5. |
- Kuipers, Grietje, et al.
(författare)
-
The tunable pReX expression vector enables optimizing the T7-based production of membrane and secretory proteins in E. coli
- 2017
-
Ingår i: Microbial Cell Factories. - : Springer Science and Business Media LLC. - 1475-2859. ; 16
-
Tidskriftsartikel (refereegranskat)abstract
- Background: To optimize the production of membrane and secretory proteins in Escherichia coli, it is critical to harmonize the expression rates of the genes encoding these proteins with the capacity of their biogenesis machineries. Therefore, we engineered the Lemo21(DE3) strain, which is derived from the T7 RNA polymerase-based BL21(DE3) protein production strain. In Lemo21(DE3), the T7 RNA polymerase activity can be modulated by the controlled co-production of its natural inhibitor T7 lysozyme. This setup enables to precisely tune target gene expression rates in Lemo21(DE3). The t7lys gene is expressed from the pLemo plasmid using the titratable rhamnose promoter. A disadvantage of the Lemo21(DE3) setup is that the system is based on two plasmids, a T7 expression vector and pLemo. The aim of this study was to simplify the Lemo21(DE3) setup by incorporating the key elements of pLemo in a standard T7-based expression vector.Results: By incorporating the gene encoding the T7 lysozyme under control of the rhamnose promoter in a standard T7-based expression vector, pReX was created (ReX stands for Regulated gene eXpression). For two model membrane proteins and a model secretory protein we show that the optimized production yields obtained with the pReX expression vector in BL21(DE3) are similar to the ones obtained with Lemo21(DE3) using a standard T7 expression vector. For another secretory protein, a c-type cytochrome, we show that pReX, in contrast to Lemo21(DE3), enables the use of a helper plasmid that is required for the maturation and hence the production of this heme c protein.Conclusions: Here, we created pReX, a T7-based expression vector that contains the gene encoding the T7 lysozyme under control of the rhamnose promoter. pReX enables regulated T7-based target gene expression using only one plasmid. We show that with pReX the production of membrane and secretory proteins can be readily optimized. Importantly, pReX facilitates the use of helper plasmids. Furthermore, the use of pReX is not restricted to BL21(DE3), but it can in principle be used in any T7 RNAP-based strain. Thus, pReX is a versatile alternative to Lemo21(DE3).
|
|
| 6. |
- Sandhu, Hena, et al.
(författare)
-
Cotranslational Translocation and Folding of a Periplasmic Protein Domain in Escherichia coli
- 2021
-
Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 0022-2836 .- 1089-8638. ; 433:15
-
Tidskriftsartikel (refereegranskat)abstract
- In Gram-negative bacteria, periplasmic domains in inner membrane proteins are cotranslationally translocated across the inner membrane through the SecYEG translocon. To what degree such domains also start to fold cotranslationally is generally difficult to determine using currently available methods. Here, we apply Force Profile Analysis (FPA) - a method where a translational arrest peptide is used to detect folding-induced forces acting on the nascent polypeptide - to follow the cotranslational translocation and folding of the large periplasmic domain of the E. coli inner membrane protease LepB in vivo. Membrane insertion of LepB's two N-terminal transmembrane helices is initiated when their respective N-terminal ends reach 45-50 residues away from the peptidyl transferase center (PTC) in the ribosome. The main folding transition in the periplasmic domain involves all but the similar to 15 most C-terminal residues of the protein and happens when the C-terminal end of the folded part is similar to 70 residues away from the PTC; a smaller putative folding intermediate is also detected. This implies that wildtype LepB folds post-translationally in vivo, and shows that FPA can be used to study both co- and post-translational protein folding in the periplasm.
|
|
