A model study of protein nascent chain and cotranslational folding using hydrophobic-polar residues

2007 ◽  
Vol 70 (2) ◽  
pp. 442-449 ◽  
Author(s):  
Hsiao-Mei Lu ◽  
Jie Liang
Keyword(s):  
Model Study ◽  
Cotranslational Folding ◽  
Polar Residues ◽  
Nascent Chain

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 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 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 Coordination of -1 Programmed Ribosomal Frameshifting by Transcript and Nascent Chain Features Revealed by Deep Mutational Scanning

2021 ◽  
Author(s):  
Patrick J. Carmody ◽  
Matthew H. Zimmer ◽  
Charles P. Kuntz ◽  
Haley R. Harrington ◽  
Kate E. Duckworth ◽  
...  
Keyword(s):  
Polypeptide Chain ◽  
Conformational Transition ◽  
Stem Loop ◽  
Ribosomal Frameshifting ◽  
Cotranslational Folding ◽  
Nascent Chain ◽  
Single Transcript ◽  
Dynamics Simulations ◽  
Mutagenic Effects ◽  
Slippery Sequence

SummaryProgrammed ribosomal frameshifting (PRF) is a translational recoding mechanism that enables the synthesis of multiple polypeptides from a single transcript. In the alphavirus structural polyprotein, -1PRF is coordinated by a “slippery” sequence in the transcript, an RNA stem-loop, and a conformational transition in the nascent polypeptide chain. To characterize each of these effectors, we measured the effects of 4,530 mutations on -1PRF by deep mutational scanning. While most mutations within the slip-site and stem-loop disrupt -1PRF, mutagenic effects upstream of the slip-site are far more variable. Molecular dynamics simulations of polyprotein biogenesis suggest many of these mutations alter stimulatory forces on the nascent chain through their effects on translocon-mediated cotranslational folding. Finally, we provide evidence suggesting the coupling between cotranslational folding and -1PRF depends on the translation kinetics upstream of the slip-site. These findings demonstrate how -1PRF is coordinated by features within both the transcript and nascent chain.

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 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 Effects of protein size, thermodynamic stability, and net charge on cotranslational folding on the ribosome

2018 ◽  
Author(s):  
José Arcadio Farías-Rico ◽  
Frida Ruud Selin ◽  
Ioanna Myronidi ◽  
Marie Frühauf ◽  
Gunnar von Heijne
Keyword(s):  
Protein Folding ◽  
Thermodynamic Stability ◽  
Protein Size ◽  
Ribosomal Protein S6 ◽  
Complex Process ◽  
Cotranslational Folding ◽  
Nascent Chain ◽  
Comprehensive Picture ◽  
Pulse Proteolysis ◽  
Net Charges

AbstractDuring the last five decades, studies of protein folding in dilute buffer solutions have produced a rich picture of this complex process. In the cell, however, proteins can start to fold while still attached to the ribosome (cotranslational folding) and it is not yet clear how the ribosome affects the folding of protein domains of different sizes, thermodynamic stabilities, and net charges. Here, by using arrest peptides as force sensors and on-ribosome pulse proteolysis, we provide a comprehensive picture of how the distance from the peptidyl transferase center in the ribosome at which proteins fold correlates with protein size. Moreover, an analysis of a large collection of mutants of theE. coliribosomal protein S6 shows that the force exerted on the nascent chain by protein folding varies linearly with the thermodynamic stability of the folded state, and that the ribosome environment disfavors folding of domains of high net-negative charge.

scholarly journals Translation and folding of single proteins in real time

2017 ◽  
Vol 114 (22) ◽  
pp. E4399-E4407 ◽  
Author(s):  
Florian Wruck ◽  
Alexandros Katranidis ◽  
Knud H. Nierhaus ◽  
Georg Büldt ◽  
Martin Hegner
Keyword(s):  
Protein Folding ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Real Time ◽  
Electrostatic Interactions ◽  
Tertiary Structure ◽  
Chain Elongation ◽  
Ribosomal Tunnel ◽  
Cotranslational Folding ◽  
Nascent Chain

Protein biosynthesis is inherently coupled to cotranslational protein folding. Folding of the nascent chain already occurs during synthesis and is mediated by spatial constraints imposed by the ribosomal exit tunnel as well as self-interactions. The polypeptide’s vectorial emergence from the ribosomal tunnel establishes the possible folding pathways leading to its native tertiary structure. How cotranslational protein folding and the rate of synthesis are linked to a protein’s amino acid sequence is still not well defined. Here, we follow synthesis by individual ribosomes using dual-trap optical tweezers and observe simultaneous folding of the nascent polypeptide chain in real time. We show that observed stalling during translation correlates with slowed peptide bond formation at successive proline sequence positions and electrostatic interactions between positively charged amino acids and the ribosomal tunnel. We also determine possible cotranslational folding sites initiated by hydrophobic collapse for an unstructured and two globular proteins while directly measuring initial cotranslational folding forces. Our study elucidates the intricate relationship among a protein’s amino acid sequence, its cotranslational nascent-chain elongation rate, and folding.

scholarly journals Effects of protein size, thermodynamic stability, and net charge on cotranslational folding on the ribosome

2018 ◽  
Vol 115 (40) ◽  
pp. E9280-E9287 ◽  
Author(s):  
José Arcadio Farías-Rico ◽  
Frida Ruud Selin ◽  
Ioanna Myronidi ◽  
Marie Frühauf ◽  
Gunnar von Heijne
Keyword(s):  
Protein Folding ◽  
Thermodynamic Stability ◽  
Protein Size ◽  
Ribosomal Protein S6 ◽  
Complex Process ◽  
Cotranslational Folding ◽  
Nascent Chain ◽  
Comprehensive Picture ◽  
Pulse Proteolysis ◽  
Net Charges

During the last five decades, studies of protein folding in dilute buffer solutions have produced a rich picture of this complex process. In the cell, however, proteins can start to fold while still attached to the ribosome (cotranslational folding) and it is not yet clear how the ribosome affects the folding of protein domains of different sizes, thermodynamic stabilities, and net charges. Here, by using arrest peptides as force sensors and on-ribosome pulse proteolysis, we provide a comprehensive picture of how the distance from the peptidyl transferase center in the ribosome at which proteins fold correlates with protein size. Moreover, an analysis of a large collection of mutants of theEscherichia coliribosomal protein S6 shows that the force exerted on the nascent chain by protein folding varies linearly with the thermodynamic stability of the folded state, and that the ribosome environment disfavors folding of domains of high net-negative charge.

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