Aarhus University Seal / Aarhus Universitets segl

Daniel Otzen

Quantitating denaturation by formic acid: Imperfect repeats are essential to the stability of the functional amyloid protein FapC

Research output: Contribution to journal/Conference contribution in journal/Contribution to newspaperJournal articleResearchpeer-review

Standard

Quantitating denaturation by formic acid : Imperfect repeats are essential to the stability of the functional amyloid protein FapC. / Christensen, Line Friis Bakmann; Nowak, Jan Stanislaw; Sønderby, Thorbjørn Vincent; Frank, Signe Andrea; Otzen, Daniel Erik.

In: The Journal of biological chemistry, Vol. 295, No. 37, 09.2020, p. 13031–13046.

Research output: Contribution to journal/Conference contribution in journal/Contribution to newspaperJournal articleResearchpeer-review

Harvard

APA

CBE

MLA

Vancouver

Author

Christensen, Line Friis Bakmann ; Nowak, Jan Stanislaw ; Sønderby, Thorbjørn Vincent ; Frank, Signe Andrea ; Otzen, Daniel Erik. / Quantitating denaturation by formic acid : Imperfect repeats are essential to the stability of the functional amyloid protein FapC. In: The Journal of biological chemistry. 2020 ; Vol. 295, No. 37. pp. 13031–13046.

Bibtex

@article{b231a3c1429d458188ae10f3e85f5679,
title = "Quantitating denaturation by formic acid: Imperfect repeats are essential to the stability of the functional amyloid protein FapC",
abstract = "Bacterial functional amyloids are evolutionarily optimized to aggregate, so much so that the extreme robustness of functional amyloid makes it very difficult to examine their structure-function relationships in a detailed manner. Previous work has shown that functional amyloids are resistant to conventional chemical denaturants, but they dissolve in formic acid (FA) at high concentrations. However, systematic investigation requires a quantitative analysis of FA's ability to denature proteins. Amyloid formed by Pseudomonas sp. protein FapC provides an excellent model to investigate FA denaturation. It contains three imperfect repeats, and stepwise removal of these repeats slows fibrillation and increases fragmentation during aggregation. However, the link to stability is unclear. We first calibrated FA denaturation using three small, globular, and acid-resistant proteins. This revealed a linear relationship between the concentration of FA and the free energy of unfolding with a slope of m FA+pH (the combined contribution of FA and FA-induced lowering of pH), as well as a robust correlation between protein size and m FA+pH We then measured the solubilization of fibrils formed from different FapC variants with varying numbers of repeats as a function of the concentration of FA. This revealed a decline in the number of residues driving amyloid formation upon deleting at least two repeats. The midpoint of denaturation declined with the removal of repeats. Complete removal of all repeats led to fibrils that were solubilized at FA concentrations 2-3 orders of magnitude lower than the repeat-containing variants, showing that at least one repeat is required for the stability of functional amyloid. ",
author = "Christensen, {Line Friis Bakmann} and Nowak, {Jan Stanislaw} and S{\o}nderby, {Thorbj{\o}rn Vincent} and Frank, {Signe Andrea} and Otzen, {Daniel Erik}",
note = "Published under license by The American Society for Biochemistry and Molecular Biology, Inc.",
year = "2020",
month = sep,
doi = "10.1074/jbc.RA120.013396",
language = "English",
volume = "295",
pages = "13031–13046",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology, Inc.",
number = "37",

}

RIS

TY - JOUR

T1 - Quantitating denaturation by formic acid

T2 - Imperfect repeats are essential to the stability of the functional amyloid protein FapC

AU - Christensen, Line Friis Bakmann

AU - Nowak, Jan Stanislaw

AU - Sønderby, Thorbjørn Vincent

AU - Frank, Signe Andrea

AU - Otzen, Daniel Erik

N1 - Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

PY - 2020/9

Y1 - 2020/9

N2 - Bacterial functional amyloids are evolutionarily optimized to aggregate, so much so that the extreme robustness of functional amyloid makes it very difficult to examine their structure-function relationships in a detailed manner. Previous work has shown that functional amyloids are resistant to conventional chemical denaturants, but they dissolve in formic acid (FA) at high concentrations. However, systematic investigation requires a quantitative analysis of FA's ability to denature proteins. Amyloid formed by Pseudomonas sp. protein FapC provides an excellent model to investigate FA denaturation. It contains three imperfect repeats, and stepwise removal of these repeats slows fibrillation and increases fragmentation during aggregation. However, the link to stability is unclear. We first calibrated FA denaturation using three small, globular, and acid-resistant proteins. This revealed a linear relationship between the concentration of FA and the free energy of unfolding with a slope of m FA+pH (the combined contribution of FA and FA-induced lowering of pH), as well as a robust correlation between protein size and m FA+pH We then measured the solubilization of fibrils formed from different FapC variants with varying numbers of repeats as a function of the concentration of FA. This revealed a decline in the number of residues driving amyloid formation upon deleting at least two repeats. The midpoint of denaturation declined with the removal of repeats. Complete removal of all repeats led to fibrils that were solubilized at FA concentrations 2-3 orders of magnitude lower than the repeat-containing variants, showing that at least one repeat is required for the stability of functional amyloid.

AB - Bacterial functional amyloids are evolutionarily optimized to aggregate, so much so that the extreme robustness of functional amyloid makes it very difficult to examine their structure-function relationships in a detailed manner. Previous work has shown that functional amyloids are resistant to conventional chemical denaturants, but they dissolve in formic acid (FA) at high concentrations. However, systematic investigation requires a quantitative analysis of FA's ability to denature proteins. Amyloid formed by Pseudomonas sp. protein FapC provides an excellent model to investigate FA denaturation. It contains three imperfect repeats, and stepwise removal of these repeats slows fibrillation and increases fragmentation during aggregation. However, the link to stability is unclear. We first calibrated FA denaturation using three small, globular, and acid-resistant proteins. This revealed a linear relationship between the concentration of FA and the free energy of unfolding with a slope of m FA+pH (the combined contribution of FA and FA-induced lowering of pH), as well as a robust correlation between protein size and m FA+pH We then measured the solubilization of fibrils formed from different FapC variants with varying numbers of repeats as a function of the concentration of FA. This revealed a decline in the number of residues driving amyloid formation upon deleting at least two repeats. The midpoint of denaturation declined with the removal of repeats. Complete removal of all repeats led to fibrils that were solubilized at FA concentrations 2-3 orders of magnitude lower than the repeat-containing variants, showing that at least one repeat is required for the stability of functional amyloid.

U2 - 10.1074/jbc.RA120.013396

DO - 10.1074/jbc.RA120.013396

M3 - Journal article

C2 - 32719003

VL - 295

SP - 13031

EP - 13046

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 37

ER -