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

Kemp, Grant; Kudva, Renuka; Rosa, Andrés de la; Heijne, Gunnar von

doi:10.1101/470831

Cited by 12 publications

(42 citation statements)

References 44 publications

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“…Region III at aa 42-52 ( Fig. 1c,d) broadly coincides with the compacted intermediate identified by FRET and FPA studies 6,7,31 . The tension increases as the entire H3 emerges below the constriction and most likely corresponds to the formation of H3 and its docking onto the preceding two-helix structure.…”

Section: Sequential Folding Of Hemk Ntd On the Ribosomesupporting

confidence: 61%

“…1d). Our results show that folding of HemK NTD is sequential 7 and that there are several tension-generating steps corresponding to folding intermediates inside and outside of the ribosome 31 . The formation of individual α-helices and tertiary interactions between α-helical elements within the exit tunnel are well documented 9,12,14,24,42 .…”

Section: -Fold Slower In C Compared To D In Different Rncs Likewismentioning

confidence: 73%

“…A high-tension mechanical pulling force (7 pN on average) originating from nascent protein folding 29 , can alleviate the SecM stalling and restart translation 14,26,29 . This phenomenon is utilized in arrest peptide-mediated force profile assays (FPA) to monitor force generation events at various stages of cotranslational folding 14,24,26,[29][30][31] . FPA can identify folding events at single-codon resolution, but this potential has not been utilized so far in identifying early folding intermediates inside the peptide exit tunnel.…”

mentioning

confidence: 99%

“…In this work, we apply FPA and PET-FCS to investigate the folding trajectory and conformational fluctuations of the α-helical N-terminal domain (NTD) of an E. coli (N5)glutamine methyltransferase protein HemK on the ribosome. We have chosen the NTD as a model protein because in solution it folds independently of the C-terminal domain and its folding on the ribosome differs dramatically from the two-state concerted folding of the free protein in solution 6,7,31 . The cotranslational folding pathway is not known in detail, but FRET and PET studies suggest that cotranslational folding of HemK NTD proceeds sequentially through several compact intermediates 6,7 , consistent with initial FPA results 31 .…”

mentioning

confidence: 99%

“…We have chosen the NTD as a model protein because in solution it folds independently of the C-terminal domain and its folding on the ribosome differs dramatically from the two-state concerted folding of the free protein in solution 6,7,31 . The cotranslational folding pathway is not known in detail, but FRET and PET studies suggest that cotranslational folding of HemK NTD proceeds sequentially through several compact intermediates 6,7 , consistent with initial FPA results 31 . Furthermore, previous time-resolved experiments showed that the observed folding dynamics is rapid compared to the pace of translation, and that the nascent chains arrested at different stages of translation (stalled RNCs) faithfully reflect the dynamics that occurs during synthesis 6 .…”

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confidence: 99%

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

Liutkute

Maiti

Samatova

et al. 2020

Preprint

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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.

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Section: Sequential Folding Of Hemk Ntd On the Ribosomesupporting

confidence: 61%

Section: -Fold Slower In C Compared To D In Different Rncs Likewismentioning

confidence: 73%

mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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

Liutkute

Maiti

Samatova

et al. 2020

Preprint

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Cotranslational folding of alkaline phosphatase in the periplasm of Escherichia coli

et al. 2020

Self Cite

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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 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 Escherichia 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.

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Slowest-first protein translation scheme: Structural asymmetry and co-translational folding

McBride

Tlusty

2021

Biophysical Journal

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Proteins are translated from the N to the C terminus, raising the basic question of how this innate directionality affects their evolution. To explore this question, we analyze 16,200 structures from the Protein Data Bank (PDB). We find remarkable enrichment of a helices at the C terminus and b strands at the N terminus. Furthermore, this a À b asymmetry correlates with sequence length and contact order, both determinants of folding rate, hinting at possible links to co-translational folding (CTF). Hence, we propose the ''slowest-first'' scheme, whereby protein sequences evolved structural asymmetry to accelerate CTF: the slowest of the cooperatively folding segments are positioned near the N terminus so they have more time to fold during translation. A phenomenological model predicts that CTF can be accelerated by asymmetry in folding rate, up to double the rate, when folding time is commensurate with translation time; analysis of the PDB predicts that structural asymmetry is indeed maximal in this regime. This correspondence is greater in prokaryotes, which generally require faster protein production. Altogether, this indicates that accelerating CTF is a substantial evolutionary force whose interplay with stability and functionality is encoded in secondary structure asymmetry.

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

Cited by 12 publications

References 44 publications

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

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

Cotranslational folding of alkaline phosphatase in the periplasm of Escherichia coli

Slowest-first protein translation scheme: Structural asymmetry and co-translational folding

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