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Publications (9 of 9) Show all publications
Sandhu, H., Hedman, R., Cymer, F., Kudva, R., Ismail, N. & von Heijne, G. (2021). Cotranslational Translocation and Folding of a Periplasmic Protein Domain in Escherichia coli. Journal of Molecular Biology, 433(15), Article ID 167047.
Open this publication in new window or tab >>Cotranslational Translocation and Folding of a Periplasmic Protein Domain in Escherichia coli
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2021 (English)In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 433, no 15, article id 167047Article in journal (Refereed) Published
Abstract [en]

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.

Keywords
cotranslational protein folding, LepB, E. coli, periplasm
National Category
Biological Sciences
Identifiers
urn:nbn:se:su:diva-197124 (URN)10.1016/j.jmb.2021.167047 (DOI)000672680300008 ()33989648 (PubMedID)
Available from: 2021-09-28 Created: 2021-09-28 Last updated: 2022-02-25Bibliographically approved
Su, T., Kudva, R., Becker, T., Buschauer, R., Komar, T., Berninghausen, O., . . . Beckmann, R. (2021). Structural basis of L-tryptophan-dependent inhibition of release factor 2 by the TnaC arrest peptide. Nucleic Acids Research, 49(16), 9539-9547
Open this publication in new window or tab >>Structural basis of L-tryptophan-dependent inhibition of release factor 2 by the TnaC arrest peptide
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2021 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 49, no 16, p. 9539-9547Article in journal (Refereed) Published
Abstract [en]

In Escherichia coli, elevated levels of free l-tryptophan (l-Trp) promote translational arrest of the TnaC peptide by inhibiting its termination. However, the mechanism by which translation-termination by the UGA-specific decoding release factor 2 (RF2) is inhibited at the UGA stop codon of stalled TnaC-ribosome-nascent chain complexes has so far been ambiguous. This study presents cryo-EM structures for ribosomes stalled by TnaC in the absence and presence of RF2 at average resolutions of 2.9 and 3.5 Å, respectively. Stalled TnaC assumes a distinct conformation composed of two small α-helices that act together with residues in the peptide exit tunnel (PET) to coordinate a single L-Trp molecule. In addition, while the peptidyl-transferase center (PTC) is locked in a conformation that allows RF2 to adopt its canonical position in the ribosome, it prevents the conserved and catalytically essential GGQ motif of RF2 from adopting its active conformation in the PTC. This explains how translation of the TnaC peptide effectively allows the ribosome to function as a L-Trp-specific small-molecule sensor that regulates the tnaCAB operon.

National Category
Biological Sciences
Identifiers
urn:nbn:se:su:diva-198856 (URN)10.1093/nar/gkab665 (DOI)000701664900039 ()34403461 (PubMedID)
Available from: 2021-11-17 Created: 2021-11-17 Last updated: 2021-11-17Bibliographically approved
Wruck, F., Tian, P., Kudva, R., Best, R. B., von Heijne, G., Tans, S. J. & Katranidis, A. (2021). The ribosome modulates folding inside the ribosomal exit tunnel. Communications Biology, 4(1), Article ID 523.
Open this publication in new window or tab >>The ribosome modulates folding inside the ribosomal exit tunnel
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2021 (English)In: Communications Biology, E-ISSN 2399-3642, Vol. 4, no 1, article id 523Article in journal (Refereed) Published
Abstract [en]

Proteins commonly fold co-translationally at the ribosome, while the nascent chain emerges from the ribosomal exit tunnel. Protein domains that are sufficiently small can even fold while still located inside the tunnel. However, the effect of the tunnel on the folding dynamics of these domains is not well understood. Here, we combine optical tweezers with single-molecule FRET and molecular dynamics simulations to investigate folding of the small zinc-finger domain ADR1a inside and at the vestibule of the ribosomal tunnel. The tunnel is found to accelerate folding and stabilize the folded state, reminiscent of the effects of chaperonins. However, a simple mechanism involving stabilization by confinement does not explain the results. Instead, it appears that electrostatic interactions between the protein and ribosome contribute to the observed folding acceleration and stabilization of ADR1a. Wruck et al. investigate the folding of the small zinc-finger domain ADR1a inside and at the vestibule of the ribosomal tunnel, using optical tweezers, single-molecule FRET, and molecular dynamics simulations. They find that the ribosomal tunnel accelerates folding while stabilizing the folded state like chaperonins. This study provides insights into the role of the ribosomal tunnel in the folding dynamics of nascent polypeptides.

National Category
Biological Sciences
Identifiers
urn:nbn:se:su:diva-195191 (URN)10.1038/s42003-021-02055-8 (DOI)000656248000007 ()33953328 (PubMedID)
Available from: 2021-08-10 Created: 2021-08-10 Last updated: 2022-02-25Bibliographically approved
Elfageih, R., Karyolaimos, A., Kemp, G., de Gier, J.-W., von Heijne, G. & Kudva, R. (2020). Cotranslational folding of alkaline phosphatase in the periplasm of Escherichia coli. Protein Science, 29(10), 2028-2037
Open this publication in new window or tab >>Cotranslational folding of alkaline phosphatase in the periplasm of Escherichia coli
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2020 (English)In: Protein Science, ISSN 0961-8368, E-ISSN 1469-896X, Vol. 29, no 10, p. 2028-2037Article in journal (Refereed) Published
Abstract [en]

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 similar to 160 angstrom, 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.

Keywords
alkaline phosphatase, disulfide bonds, force profile analysis, periplasm, protein folding
National Category
Biological Sciences
Identifiers
urn:nbn:se:su:diva-185318 (URN)10.1002/pro.3927 (DOI)000561907600001 ()32790204 (PubMedID)
Available from: 2020-11-23 Created: 2020-11-23 Last updated: 2022-02-25Bibliographically approved
Andersson, A., Kudva, R., Magoulopoulou, A., Lejarre, Q., Lara, P., Xu, P., . . . Tellgren-Roth, Å. (2020). Membrane integration and topology of RIFIN and STEVOR proteins of the Plasmodium falciparum parasite. The FEBS Journal, 287(13), 2744-2762
Open this publication in new window or tab >>Membrane integration and topology of RIFIN and STEVOR proteins of the Plasmodium falciparum parasite
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2020 (English)In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 287, no 13, p. 2744-2762Article in journal (Refereed) Published
Abstract [en]

The malarial parasite Plasmodium exports its own proteins to the cell surfaces of red blood cells (RBCs) during infection. Examples of exported proteins include members of the repetitive interspersed family (RIFIN) and subtelomeric variable open reading frame (STEVOR) family of proteins from Plasmodium falciparum. The presence of these parasite-derived proteins on surfaces of infected RBCs triggers the adhesion of infected cells to uninfected cells (rosetting) and to the vascular endothelium potentially obstructing blood flow. While there is a fair amount of information on the localization of these proteins on the cell surfaces of RBCs, less is known about how they can be exported to the membrane and the topologies they can adopt during the process. The first step of export is plausibly the cotranslational insertion of proteins into the endoplasmic reticulum (ER) of the parasite, and here, we investigate the insertion of three RIFIN and two STEVOR proteins into the ER membrane. We employ a well-established experimental system that uses N-linked glycosylation of sites within the protein as a measure to assess the extent of membrane insertion and the topology it assumes when inserted into the ER membrane. Our results indicate that for all the proteins tested, transmembranes (TMs) 1 and 3 integrate into the membrane, so that the protein assumes an overall topology of Ncyt-Ccyt. We also show that the segment predicted to be TM2 for each of the proteins likely does not reside in the membrane, but is translocated to the lumen.

Keywords
membrane protein topology, N-linked glycosylation, Plasmodium, RIFIN protein, STEVOR protein
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:su:diva-177797 (URN)10.1111/febs.15171 (DOI)000504344200001 ()31821735 (PubMedID)
Available from: 2020-01-21 Created: 2020-01-21 Last updated: 2022-02-26Bibliographically approved
Kemp, G., Kudva, R., de la Rosa, A. & von Heijne, G. (2019). Force-Profile Analysis of the Cotranslational Folding of HemK and Filamin Domains: Comparison of Biochemical and Biophysical Folding Assays. Journal of Molecular Biology, 431(6), 1308-1314
Open this publication in new window or tab >>Force-Profile Analysis of the Cotranslational Folding of HemK and Filamin Domains: Comparison of Biochemical and Biophysical Folding Assays
2019 (English)In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 431, no 6, p. 1308-1314Article in journal (Refereed) Published
Abstract [en]

We have characterized the cotranslational folding of two small protein domains of different folds-the alpha-helical N-terminal domain of HemK and the beta-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, or FPA), allowing us to compare FPA to three other techniques currently used to study cotranslational folding: real-time FRET, photo induced electron transfer, 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.

Keywords
cotranslational folding, HemK, FLN5, arrest peptide
National Category
Biological Sciences
Identifiers
urn:nbn:se:su:diva-168378 (URN)10.1016/j.jmb.2019.01.043 (DOI)000463120800018 ()30738895 (PubMedID)
Available from: 2019-05-06 Created: 2019-05-06 Last updated: 2022-02-26Bibliographically approved
Tian, P., Steward, A., Kudva, R., Su, T., Shilling, P. J., Nickson, A. A., . . . Best, R. B. (2018). Folding pathway of an Ig domain is conserved on and off the ribosome. Proceedings of the National Academy of Sciences of the United States of America, 115(48), E11284-E11293
Open this publication in new window or tab >>Folding pathway of an Ig domain is conserved on and off the ribosome
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2018 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 115, no 48, p. E11284-E11293Article in journal (Refereed) Published
Abstract [en]

Proteins that fold cotranslationally may do so in a restricted configurational space, due to the volume occupied by the ribosome. How does this environment, coupled with the close proximity of the ribosome, affect the folding pathway of a protein? Previous studies have shown that the cotranslational folding process for many proteins, including small, single domains, is directly affected by the ribosome. Here, we investigate the cotranslational folding of an all-beta Ig domain, titin I27. Using an arrest peptide-based assay and structural studies by cryo-EM, we show that I27 folds in the mouth of the ribosome exit tunnel. Simulations that use a kinetic model for the force dependence of escape from arrest accurately predict the fraction of folded protein as a function of length. We used these simulations to probe the folding pathway on and off the ribosome. Our simulations-which also reproduce experiments on mutant forms of I27-show that I27 folds, while still sequestered in the mouth of the ribosome exit tunnel, by essentially the same pathway as free I27, with only subtle shifts of critical contacts from the C to the N terminus.

Keywords
arrest peptide, fraction folded, mechanical force, molecular simulation, kinetic model
National Category
Biological Sciences
Identifiers
urn:nbn:se:su:diva-162838 (URN)10.1073/pnas.1810523115 (DOI)000451351000015 ()30413621 (PubMedID)
Available from: 2018-12-10 Created: 2018-12-10 Last updated: 2022-03-23Bibliographically approved
Kudva, R., Tian, P., Pardo-Avila, F., Carroni, M., Best, R. B., Bernstein, H. D. & von Heijne, G. (2018). The shape of the bacterial ribosome exit tunnel affects cotranslational protein folding. eLIFE, 7, Article ID e36326.
Open this publication in new window or tab >>The shape of the bacterial ribosome exit tunnel affects cotranslational protein folding
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2018 (English)In: eLIFE, E-ISSN 2050-084X, Vol. 7, article id e36326Article in journal (Refereed) Published
Abstract [en]

The E. coli ribosome exit tunnel can accommodate small folded proteins, while larger ones fold outside. It remains unclear, however, to what extent the geometry of the tunnel influences protein folding. Here, using E. coli ribosomes with deletions in loops in proteins uL23 and uL24 that protrude into the tunnel, we investigate how tunnel geometry determines where proteins of different sizes fold. We find that a 29-residue zinc-finger domain normally folding close to the uL23 loop folds deeper in the tunnel in uL23 Delta loop ribosomes, while two similar to 100 residue proteins normally folding close to the uL24 loop near the tunnel exit port fold at deeper locations in uL24 Delta loop ribosomes, in good agreement with results obtained by coarse-grained molecular dynamics simulations. This supports the idea that cotranslational folding commences once a protein domain reaches a location in the exit tunnel where there is sufficient space to house the folded structure.

National Category
Biological Sciences
Identifiers
urn:nbn:se:su:diva-163616 (URN)10.7554/eLife.36326 (DOI)000453505600001 ()30475203 (PubMedID)
Available from: 2019-01-08 Created: 2019-01-08 Last updated: 2022-03-23Bibliographically approved
Kudva, R. & Götzke, H.YfgM plays a role in protein insertion through the SecYEG translocon.
Open this publication in new window or tab >>YfgM plays a role in protein insertion through the SecYEG translocon
(English)Manuscript (preprint) (Other academic)
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-106274 (URN)
Available from: 2014-07-29 Created: 2014-07-29 Last updated: 2022-02-23Bibliographically approved
Organisations
Identifiers
ORCID iD: ORCID iD iconorcid.org/0000-0003-0426-3716

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