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 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 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 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

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 Cotranslational folding allows misfolding-prone proteins to circumvent deep kinetic traps

2020 ◽  
Vol 117 (3) ◽  
pp. 1485-1495 ◽  
Author(s):  
Amir Bitran ◽  
William M. Jacobs ◽  
Xiadi Zhai ◽  
Eugene Shakhnovich
Keyword(s):  
Self Assembly ◽  
Molecular Mechanisms ◽  
Folding Kinetics ◽  
Rare Codons ◽  
Cotranslational Folding ◽  
Nascent Chain ◽  
Kinetic Traps ◽  
Optimal Window

Many large proteins suffer from slow or inefficient folding in vitro. It has long been known that this problem can be alleviated in vivo if proteins start folding cotranslationally. However, the molecular mechanisms underlying this improvement have not been well established. To address this question, we use an all-atom simulation-based algorithm to compute the folding properties of various large protein domains as a function of nascent chain length. We find that for certain proteins, there exists a narrow window of lengths that confers both thermodynamic stability and fast folding kinetics. Beyond these lengths, folding is drastically slowed by nonnative interactions involving C-terminal residues. Thus, cotranslational folding is predicted to be beneficial because it allows proteins to take advantage of this optimal window of lengths and thus avoid kinetic traps. Interestingly, many of these proteins’ sequences contain conserved rare codons that may slow down synthesis at this optimal window, suggesting that synthesis rates may be evolutionarily tuned to optimize folding. Using kinetic modeling, we show that under certain conditions, such a slowdown indeed improves cotranslational folding efficiency by giving these nascent chains more time to fold. In contrast, other proteins are predicted not to benefit from cotranslational folding due to a lack of significant nonnative interactions, and indeed these proteins’ sequences lack conserved C-terminal rare codons. Together, these results shed light on the factors that promote proper protein folding in the cell and how biomolecular self-assembly may be optimized evolutionarily.

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 Cotranslational protein folding can promote the formation of correct folding intermediate

2020 ◽  
Author(s):  
P. Tao ◽  
Y. Xiao
Keyword(s):  
Molecular Dynamics ◽  
Protein Folding ◽  
Molecular Dynamics Simulations ◽  
General Scheme ◽  
Folding Intermediate ◽  
Microscopic Mechanism ◽  
Cotranslational Folding ◽  
Exit Tunnel ◽  
Dynamics Simulations

AbstractCotranslational folding is vital for proteins to form correct structures in vivo. However, it is still unclear how a nascent chain folds at atomic resolution during the translation process. Previously, we have built a model of ribosomal exit tunnel and investigated cotranslational folding of a three-helices protein by using all-atom molecular dynamics simulations. Here we shall study the cotranslational folding of three mainly-β proteins using the same method and find that cotranslational folding can enhance helical population in most cases and reduce nonnative long-range contacts before emerging from the ribosomal exit tunnel. After exiting the tunnel, all proteins fall into local minimal states and structural ensembles in cotranslational folding are more helical than in free folding. Importantly, for GTT WW domain, one local minimal state in cotranslational folding is known as correct folding intermediate, which is not found in free folding. This result suggests that cotranslational folding may directly increase folding efficiency by accelerating sampling more than by avoiding the misfolded state, which is a mainstream viewpoint in present. In addition, our method can serve as a general scheme to study cotranslational folding process of proteins.Statement of SignificanceIn cell, the formations of correct three-dimensional structures of proteins, namely protein folding, are essential to human health. Misfolding can lead to serious diseases such as Alzheimer’s disease and mad cow disease. As the first step of in vivo folding, the effect of cotranslational folding on the correct folding of proteins has been the focus of scientific research in this century. Although some experiments have shown that cotranslational folding can improve the efficiency of folding, its microscopic mechanism is not yet clear. In this paper, we study the process of cotranslational folding of three proteins by using all-atom molecular dynamics simulations, and try to reveal some aspects of the mechanism of cotranslational folding from a microscopic perspective.

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.

Sign in / Sign up

Export Citation Format

Share Document