scholarly journals Cotranslational folding of alkaline phosphatase in the periplasm of Escherichia coli

2020 ◽  
Author(s):  
Rageia Elfageih ◽  
Alexandros Karyolaimos ◽  
Grant Kemp ◽  
Jan-Willem de Gier ◽  
Gunnar von Heijne ◽  
...  
Keyword(s):  
Alkaline Phosphatase ◽  
Wild Type Strain ◽  
Profile Analysis ◽  
E Coli ◽  
Close Proximity ◽  
Cotranslational Folding ◽  
Nascent Chain ◽  
Force Profile ◽  
Translational Arrest ◽  
Strain Background

AbstractCotranslational 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.

scholarly journals Cotranslational folding of a periplasmic protein domain in Escherichia coli

2021 ◽  
Author(s):  
Hena Sandhu ◽  
Rickard Hedman ◽  
Florian Cymer ◽  
Renuka Kudva ◽  
Nurzian Ismail ◽  
...  
Keyword(s):  
Profile Analysis ◽  
Protein Domain ◽  
Transmembrane Helices ◽  
Inner Membrane ◽  
E Coli ◽  
Cotranslational Folding ◽  
Force Profile ◽  
Translational Arrest ◽  
Periplasmic Domain

AbstractIn 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 ~15 most C-terminal residues of the protein and happens when the C-terminal end of the folded part is ~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.

scholarly journals Gradual Compaction of the Nascent Peptide During Cotranslational Folding on the Ribosome

2020 ◽  
Author(s):  
Marija Liutkute ◽  
Manisankar Maiti ◽  
Ekaterina Samatova ◽  
Jörg Enderlein ◽  
Marina V. Rodnina
Keyword(s):  
Profile Analysis ◽  
Dynamic Properties ◽  
Correlation Spectroscopy ◽  
Fluorescence Correlation ◽  
Cotranslational Folding ◽  
Nascent Peptide ◽  
Nascent Chain ◽  
Force Profile ◽  
Exit Tunnel ◽  
Domain Stabilization

ABSTRACTNascent polypeptides begin to fold in the constrained space of the ribosomal peptide exit tunnel. Here we use force profile analysis (FPA) and photo-induced energy-transfer fluorescence correlation spectroscopy (PET-FCS) to show how a small α-helical domain, the N-terminal domain of HemK, folds cotranslationally. Compaction starts vectorially as soon as the first α-helical segments are synthesized. As nascent chain grows, emerging helical segments dock onto each other and continue to rearrange at the vicinity of the ribosome. Inside or in the proximity of the ribosome, the nascent peptide undergoes structural fluctuations on the μs time scale. The fluctuations slow down as the domain moves away from the ribosome. Folding mutations have little effect on folding within the exit tunnel, but abolish the final domain stabilization. The results show the power of FPA and PET-FCS in solving the trajectory of cotranslational protein folding and in characterizing the dynamic properties of folding intermediates.

scholarly journals Cotranslational folding cooperativity of contiguous domains of α-spectrin

2019 ◽  
Author(s):  
Grant Kemp ◽  
Ola B. Nilsson ◽  
Pengfei Tian ◽  
Robert B. Best ◽  
Gunnar von Heijne
Keyword(s):  
Profile Analysis ◽  
Full Length ◽  
Protein Chain ◽  
Multidomain Proteins ◽  
Cotranslational Folding ◽  
Nascent Chain ◽  
Force Profile ◽  
Biochemical Measurements ◽  
Dynamics Simulations

AbstractProteins synthesized in the cell can begin to fold during translation before the entire polypeptide has been produced, which may be particularly relevant to the folding of multidomain proteins. Here, we study the cotranslational folding of adjacent domains from the cytoskeletal protein α-spectrin using Force Profile Analysis (FPA). Specifically, we investigate how the cotranslational folding behavior of the R15 and R16 domains are affected by their neighboring R14 and R16, and R15 and R17 domains, respectively. Our results show that the domains impact each other’s folding in distinct ways that may be important for the efficient assembly of α-spectrin, and may reduce its dependence on chaperones. Furthermore, we directly relate the experimentally observed yield of full-length protein in the FPA assay to the force exerted by the folding protein in pN. By combining pulse-chase experiments to measure the rate at which the arrested protein is converted into full-length protein with a Bell model of force-induced rupture, we estimate that the R16 domain exerts a maximal force on the nascent chain of ∼15 pN during cotranslational folding.SignificanceIn living cells, proteins are produced in a sequential way by ribosomes. This vectoral process allows the growing protein chain to start to fold before translation has been completed. Thereby, cotranslational protein folding can be significantly different than the folding of a full-length protein in isolation. Here we show how structurally similar repeat domains, normally produced as parts of a single long polypeptide, affect the cotranslational folding of their neighbors. This provides insight into how the cell may efficiently produce multidomain proteins, paving the way for future studies in vivo or with chaperones. We also provide an estimated magnitude of the mechanical force on the nascent chain generated by cotranslational folding, calculated from biochemical measurements and molecular dynamics simulations.

scholarly journals Cotranslational folding cooperativity of contiguous domains of α-spectrin

2020 ◽  
Vol 117 (25) ◽  
pp. 14119-14126 ◽  
Author(s):  
Grant Kemp ◽  
Ola B. Nilsson ◽  
Pengfei Tian ◽  
Robert B. Best ◽  
Gunnar von Heijne
Keyword(s):  
Profile Analysis ◽  
Full Length ◽  
Cytoskeletal Protein ◽  
Multidomain Proteins ◽  
Maximal Force ◽  
Cotranslational Folding ◽  
Nascent Chain ◽  
Force Profile ◽  
Bell Model

Proteins synthesized in the cell can begin to fold during translation before the entire polypeptide has been produced, which may be particularly relevant to the folding of multidomain proteins. Here, we study the cotranslational folding of adjacent domains from the cytoskeletal protein α-spectrin using force profile analysis (FPA). Specifically, we investigate how the cotranslational folding behavior of the R15 and R16 domains are affected by their neighboring R14 and R16, and R15 and R17 domains, respectively. Our results show that the domains impact each other’s folding in distinct ways that may be important for the efficient assembly of α-spectrin, and may reduce its dependence on chaperones. Furthermore, we directly relate the experimentally observed yield of full-length protein in the FPA assay to the force exerted by the folding protein in piconewtons. By combining pulse-chase experiments to measure the rate at which the arrested protein is converted into full-length protein with a Bell model of force-induced rupture, we estimate that the R16 domain exerts a maximal force on the nascent chain of ∼15 pN during cotranslational folding.

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

2018 ◽  
Author(s):  
Grant Kemp ◽  
Renuka Kudva ◽  
Andrés de la Rosa ◽  
Gunnar von Heijne
Keyword(s):  
Real Time ◽  
Profile Analysis ◽  
The Other ◽  
Basic Process ◽  
Cotranslational Folding ◽  
Nascent Chain ◽  
Force Profile ◽  
Exit Tunnel ◽  
Ribosome Exit Tunnel ◽  
Different Parts

AbstractWe have characterized the cotranslational folding of two small protein domains of different folds – the a-helical N-terminal domain of HemK and the β-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.

scholarly journals Gradual compaction of the nascent peptide during cotranslational folding on the ribosome

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Marija Liutkute ◽  
Manisankar Maiti ◽  
Ekaterina Samatova ◽  
Jörg Enderlein ◽  
Marina V Rodnina
Keyword(s):  
Profile Analysis ◽  
Dynamic Properties ◽  
Correlation Spectroscopy ◽  
Hydrophobic Core ◽  
Fluorescence Correlation ◽  
Nascent Peptide ◽  
Nascent Chain ◽  
Force Profile ◽  
Exit Tunnel ◽  
Domain Stabilization

Nascent polypeptides begin to fold in the constrained space of the ribosomal peptide exit tunnel. Here we use force-profile analysis (FPA) and photo-induced energy-transfer fluorescence correlation spectroscopy (PET-FCS) to show how a small α-helical domain, the N-terminal domain of HemK, folds cotranslationally. Compaction starts vectorially as soon as the first α-helical segments are synthesized. As nascent chain grows, emerging helical segments dock onto each other and continue to rearrange at the vicinity of the ribosome. Inside or in the proximity of the ribosome, the nascent peptide undergoes structural fluctuations on the µs time scale. The fluctuations slow down as the domain moves away from the ribosome. Mutations that destabilize the packing of the domain’s hydrophobic core have little effect on folding within the exit tunnel, but abolish the final domain stabilization. The results show the power of FPA and PET-FCS in solving the trajectory of cotranslational protein folding and in characterizing the dynamic properties of folding intermediates.

scholarly journals Mutational analysis of the human Xbp1 translational arrest peptide and construction of arrest-enhanced variants

2017 ◽  
Author(s):  
Nina Schiller ◽  
Anastasia Magoulopoulou ◽  
Florian Cymer ◽  
Gunnar Von Heijne
Keyword(s):  
Mutational Analysis ◽  
Strong Increase ◽  
E Coli ◽  
Unfolded Protein ◽  
Nascent Chain ◽  
Translational Arrest ◽  
Critical Residues ◽  
The Unfolded Protein Response ◽  
Natural Amino Acids ◽  
Protein Response

AbstractXbp1, a protein involved in the unfolded protein response, is a rare example of a mammalian protein that contains a well-defined translational arrest peptide (AP). In order to define the critical residues in the Xbp1u AP, and to search for variants with stronger arrest potency than the wildtype Xbp1u AP, we have carried out a full mutagenesis scan where each residue in the AP was replaced by the other 19 natural amino acids. We find that 10 of the 21 mutagenized positions are optimal already in the wildtype Xbp1 AP, while certain mutations in the remaining residues lead to a strong increase in the arrest potency. Xbp1 has thus evolved to induce an intermediate level of translational arrest, and versions with much stronger arrest efficiency exist. We further show Xbp1-induced translational arrest is reduced in response to increased tension in the nascent chain, making it possible to carry out studies in mammalian systems of cotranslational processes such as membrane protein assembly and protein folding by using suitable Xbp1 AP variants as “force sensors”, as has been done previously in E. coli using bacterial APs.

scholarly journals The folding and unfolding behavior of ribonuclease H on the ribosome

2020 ◽  
Vol 295 (33) ◽  
pp. 11410-11417 ◽  
Author(s):  
Madeleine K. Jensen ◽  
Avi J. Samelson ◽  
Annette Steward ◽  
Jane Clarke ◽  
Susan Marqusee
Keyword(s):  
Protein Stability ◽  
Ribosomal Proteins ◽  
Profile Analysis ◽  
Rnase H ◽  
Folding Intermediate ◽  
Folding Rate ◽  
Nascent Chain ◽  
Force Profile ◽  
Pulse Proteolysis

The health of a cell depends on accurate translation and proper protein folding, whereas misfolding can lead to aggregation and disease. The first opportunity for a protein to fold occurs during translation, when the ribosome and surrounding environment can affect the nascent chain energy landscape. However, quantifying these environmental effects is challenging because ribosomal proteins and rRNA preclude most spectroscopic measurements of protein energetics. Here, we have applied two gel-based approaches, pulse proteolysis and force-profile analysis, to probe the folding and unfolding pathways of RNase H (RNH) nascent chains stalled on the prokaryotic ribosome in vitro. We found that ribosome-stalled RNH has an increased unfolding rate compared with free RNH. Because protein stability is related to the ratio of the unfolding and folding rates, this increase completely accounts for the observed change in protein stability and indicates that the folding rate is unchanged. Using arrest peptide–based force-profile analysis, we assayed the force generated during the folding of RNH on the ribosome. Surprisingly, we found that population of the RNH folding intermediate is required to generate sufficient force to release a stall induced by the SecM stalling sequence and that readthrough of SecM directly correlates with the stability of the RNH folding intermediate. Together, these results imply that the folding pathway of RNH is unchanged on the ribosome. Furthermore, our findings indicate that the ribosome promotes RNH unfolding while the nascent chain is proximal to the ribosome, which may limit the deleterious effects of RNH misfolding and assist in folding fidelity.

scholarly journals The folding and unfolding behavior of ribonuclease H on the ribosome

2020 ◽  
Author(s):  
Madeleine K. Jensen ◽  
Avi J. Samelson ◽  
Annette Steward ◽  
Jane Clarke ◽  
Susan Marqusee
Keyword(s):  
Ribosomal Proteins ◽  
Profile Analysis ◽  
Rnase H ◽  
Folding Intermediate ◽  
Folding Rate ◽  
Nascent Chain ◽  
Force Profile ◽  
A Cell ◽  
The Stability ◽  
Pulse Proteolysis

ABSTRACTThe health of a cell depends on accurate translation and proper protein folding; misfolding can lead to aggregation and disease. The first opportunity for a protein to fold occurs during translation, when the ribosome and surrounding environment can affect the energy landscape of the nascent chain. However, quantifying these environmental effects is challenging due to the ribosomal proteins and rRNA, which preclude most spectroscopic measurements of protein energetics. We have applied two gel-based approaches, pulse proteolysis and force-peptide arrest assays, to probe the folding and unfolding pathways of RNase H ribosome-stalled nascent chains. We find that ribosome-stalled RNase H has an increased unfolding rate compared to free RNase H, which completely accounts for observed changes in protein stability and indicates that the folding rate is unchanged. Using arrest peptide-based force-profile analysis, we assayed the force generated during the folding of RNase H on the ribosome. Surprisingly, we find that population of the RNase H folding intermediate is required to generate sufficient force to release the SecM stall and that readthrough of the stall sequence directly correlates with the stability of the folding intermediate. Together, these data imply that the folding pathway of RNase H is unchanged on the ribosome. Furthermore, our data indicate that the ribosome promotes unfolding while the nascent chain is proximal to the ribosome, which may limit the deleterious effects of misfolding and assist in folding fidelity.

scholarly journals Increased freedom of movement in the nascent chain results in dynamic changes in the structure of the SecM arrest motif

2019 ◽  
Vol 39 (1) ◽  
Author(s):  
Hazel A. Bracken ◽  
Cheryl A. Woolhead
Keyword(s):  
Point Mutations ◽  
Translation Arrest ◽  
E Coli ◽  
Freedom Of Movement ◽  
Conserved Sequence ◽  
C Terminus ◽  
Nascent Chain ◽  
Translational Arrest ◽  
Exit Tunnel ◽  
Ribosome Exit Tunnel

Abstract Ribosomes are responsible for the synthesis of all cellular proteins. Due to the diversity of sequence and properties, it was initially believed that translating nascent chains would travel unhindered through the ribosome exit tunnel, however a small but increasing number of proteins have been identified that interact with the exit tunnel to induce translational arrest, Escherichia coli (E. coli) secretion monitor (SecM) is one such stalling peptide. How and why these peptides interact with the exit tunnel is not fully understood, however key features required for stalling appear to be an essential peptide arrest motif at the C-terminus and compaction of the nascent chain within the exit tunnel upon stalling. Mutagenesis of the SecM arrest sequence has identified three conservative point mutations that can retain a degree of stalling in this highly conserved sequence. This level of stalling is further increased when coupled with mutation of a non-essential arrest motif residue P153A. Further analysis of these mutants by pegylation assays indicates that this increase in stalling activity during translation is due to the ability of the P153A mutation to reintroduce compaction of the nascent chain within the exit tunnel possibly due to the improved flexibility of the nascent chain provided by the removal of a restrictive proline residue. The data presented here suggest that arrest sequences may be more prevalent and less highly conserved than previously thought, and highlight the significance of the interactions between the nascent chain and the exit tunnel to affecting translation arrest.

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