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• W3043970664 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 mFA+pH (the combined contribution of FA and FA-induced lowering of pH), as well as a robust correlation between protein size and mFA+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. 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 mFA+pH (the combined contribution of FA and FA-induced lowering of pH), as well as a robust correlation between protein size and mFA+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. The term amyloid is normally associated with misfolding of different proteins, resulting in neurodegenerative diseases like Alzheimer's and Parkinson's disease, but the number of cases where the amyloid structure is used for functional purposes is steadily increasing (1Pham C.L. Kwan A.H. Sunde M. Functional amyloid: widespread in Nature, diverse in purpose.Essays Biochem. 2014; 56 (25131597): 207-21910.1042/bse0560207Crossref PubMed Google Scholar, 2Dueholm M. Nielsen P.H. Chapman M.R. Otzen D.E. Functional amyloids in Bacteria.in: Otzen D.E. Amyloid Fibrils and Prefibrillar Aggregates. Wiley-VCH Verlag GmbH, Berlin, Germany2012: 411-438Google Scholar). The first functional amyloids to be identified and purified were the curli fibrils expressed by Escherichia coli and Salmonella enteritidis (3Olsen A. Jonsson A. Normark S. Fibronectin binding mediated by a novel class of surface organelles on Escherichia coli.Nature. 1989; 338 (2649795): 652-65510.1038/338652a0Crossref PubMed Scopus (438) Google Scholar, 4Collinson S.K. Emody L. Muller K.H. Trust T.J. Kay W.W. Purification and characterization of thin, aggregative fimbriae from Salmonella enteritidis.J. Bacteriol. 1991; 173 (1677357): 4773-478110.1128/jb.173.15.4773-4781.1991Crossref PubMed Scopus (289) Google Scholar). Since their discovery, curli have been shown to be widely expressed among different bacteria, spanning at least four different phyla (5Dueholm M.S. Albertsen M. Otzen D. Nielsen P.H. Curli functional amyloid systems are phylogenetically widespread and display large diversity in operon and protein structure.PLoS ONE. 2012; 7 (23251478): e5127410.1371/journal.pone.0051274Crossref PubMed Scopus (90) Google Scholar). Curli fibrils serve an architectural role in bacterial biofilm but are also involved in cell attachment and invasion (6Kikuchi T. Mizunoe Y. Takade A. Naito S. Yoshida S. Curli fibers are required for development of biofilm architecture in Escherichia coli K-12 and enhance bacterial adherence to human uroepithelial cells.Microbiol. Immunol. 2005; 49 (16172544): 875-88410.1111/j.1348-0421.2005.tb03678.xCrossref PubMed Scopus (132) Google Scholar, 7Uhlich G.A. Keen J.E. Elder R.O. Variations in the csgD promoter of Escherichia coli O157:H7 associated with increased virulence in mice and increased invasion of HEp-2 cells.Infect. Immun. 2002; 70 (11748206): 395-39910.1128/iai.70.1.395-399.2002Crossref PubMed Scopus (84) Google Scholar, 8Gophna U. Barlev M. Seijffers R. Oelschlager T.A. Hacker J. Ron E.Z. Curli fibers mediate internalization of Escherichia coli by eukaryotic cells.Infect. Immun. 2001; 69 (11254632): 2659-266510.1128/IAI.69.4.2659-2665.2001Crossref PubMed Scopus (154) Google Scholar). Curli are composed primarily of the protein CsgA, which, for bacterial strains within the Enterobacteriales and the Vibrionales, is expressed from the csgBAC operon together with the nucleator protein CsgB and the chaperone CsgC (5Dueholm M.S. Albertsen M. Otzen D. Nielsen P.H. Curli functional amyloid systems are phylogenetically widespread and display large diversity in operon and protein structure.PLoS ONE. 2012; 7 (23251478): e5127410.1371/journal.pone.0051274Crossref PubMed Scopus (90) Google Scholar, 9Hammar M. Arnqvist A. Bian Z. Olsen A. Normark S. Expression of two csg operons is required for production of fibronectin- and congo red-binding curli polymers in Escherichia coli K-12.Mol. Microbiol. 1995; 18 (28776848): 661-67010.1111/j.1365-2958.1995.mmi_18040661.xCrossref PubMed Scopus (367) Google Scholar, 10Chapman M.R. Robinson L.S. Pinkner J.S. Roth R. Heuser J. Hammar M. Normark S. Hultgren S.J. Role of Escherichia coli curli operons in directing amyloid fiber formation.Science. 2002; 295 (11823641): 851-85510.1126/science.1067484Crossref PubMed Scopus (895) Google Scholar). Furthermore, four additional Csg proteins are expressed from the csgDEFG operon, and they act as a transcription regulator of the csgBAC operon (CsgD), chaperones (CsgE/CsgF), and an outer membrane pore protein (CsgG) (9Hammar M. Arnqvist A. Bian Z. Olsen A. Normark S. Expression of two csg operons is required for production of fibronectin- and congo red-binding curli polymers in Escherichia coli K-12.Mol. Microbiol. 1995; 18 (28776848): 661-67010.1111/j.1365-2958.1995.mmi_18040661.xCrossref PubMed Scopus (367) Google Scholar, 10Chapman M.R. Robinson L.S. Pinkner J.S. Roth R. Heuser J. Hammar M. Normark S. Hultgren S.J. Role of Escherichia coli curli operons in directing amyloid fiber formation.Science. 2002; 295 (11823641): 851-85510.1126/science.1067484Crossref PubMed Scopus (895) Google Scholar, 11Robinson L.S. Ashman E.M. Hultgren S.J. Chapman M.R. Secretion of curli fibre subunits is mediated by the outer membrane-localized CsgG protein.Mol. Microbiol. 2006; 59 (16420357): 870-88110.1111/j.1365-2958.2005.04997.xCrossref PubMed Scopus (162) Google Scholar). All Csg proteins, except CsgD, are targeted for Sec-dependent secretion across the inner bacterial membrane to the periplasm (12Barnhart M.M. Chapman M.R. Curli biogenesis and function.Annu. Rev. Microbiol. 2006; 60 (16704339): 131-14710.1146/annurev.micro.60.080805.142106Crossref PubMed Scopus (720) Google Scholar). A decade ago, we identified a similar amyloid system in Pseudomonas, termed functional amyloid in Pseudomonas (fap) (13Dueholm M.S. Petersen S.V. Sønderkær M. Larsen P. Christiansen G. Hein K.L. Enghild J.J. Nielsen J.L. Nielsen K.L. Nielsen P.H. Otzen D.E. Functional amyloid in Pseudomonas.Mol. Microbiol. 2010; 77 (20572935): 1009-102010.1111/j.1365-2958.2010.07269.xCrossref PubMed Scopus (188) Google Scholar). The fap system encodes proteins FapA–F, expressed from a single fapA–F operon. Like the curli system, it encodes an outer membrane pore protein (FapF) and possible chaperones (FapA and FapD), besides the primary amyloid-forming protein FapC, and a potential nucleator FapB (14Rouse S.L. Matthews S.J. Dueholm M.S. Ecology and biogenesis of functional amyloids in Pseudomonas.J. Mol. Biol. 2018; 430: 3685-369510.1016/j.jmb.2018.05.004Crossref PubMed Scopus (31) Google Scholar) (Fig. 1). Like the curli system, fap fibrils contribute to biofilm formation (15Dueholm M.S. Sondergaard M.T. Nilsson M. Christiansen G. Stensballe A. Overgaard M.T. Givskov M. Tolker-Nielsen T. Otzen D.E. Nielsen P.H. Expression of Fap amyloids in Pseudomonas aeruginosa, P. fluorescens, and P. putida results in aggregation and increased biofilm formation.Microbiologyopen. 2013; 2 (23504942): 365-38210.1002/mbo3.81Crossref PubMed Scopus (100) Google Scholar, 16Zeng G. Vad B.S. Dueholm M.S. Christiansen G. Nilsson M. Tolker-Nielsen T. Nielsen P.H. Meyer R.L. Otzen D.E. Functional bacterial amyloid increases Pseudomonas biofilm hydrophobicity and stiffness.Front. Microbiol. 2015; 6 (26500638): 109910.3389/fmicb.2015.01099Crossref PubMed Scopus (94) Google Scholar) and play roles in virulence (17Wiehlmann L. Munder A. Adams T. Juhas M. Kolmar H. Salunkhe P. Tummler B. Functional genomics of Pseudomonas aeruginosa to identify habitat-specific determinants of pathogenicity.Int J. Med. Microbiol. 2007; 297 (17481950): 615-62310.1016/j.ijmm.2007.03.014Crossref PubMed Scopus (26) Google Scholar), and the individual Fap proteins are also secreted across the inner membrane via Sec (15Dueholm M.S. Sondergaard M.T. Nilsson M. Christiansen G. Stensballe A. Overgaard M.T. Givskov M. Tolker-Nielsen T. Otzen D.E. Nielsen P.H. Expression of Fap amyloids in Pseudomonas aeruginosa, P. fluorescens, and P. putida results in aggregation and increased biofilm formation.Microbiologyopen. 2013; 2 (23504942): 365-38210.1002/mbo3.81Crossref PubMed Scopus (100) Google Scholar, 18Rouse S.L. Hawthorne W.J. Berry J.L. Chorev D.S. Ionescu S.A. Lambert S. Stylianou F. Ewert W. Mackie U. Morgan R.M.L. Otzen D. Herbst F.A. Nielsen P.H. Dueholm M. Bayley H. et al.A new class of hybrid secretion system is employed in Pseudomonas amyloid biogenesis.Nat. Commun. 2017; 8 (28811582): 26310.1038/s41467-017-00361-6Crossref PubMed Scopus (39) Google Scholar, 19Lewenza S. Gardy J.L. Brinkman F.S. Hancock R.E. Genome-wide identification of Pseudomonas aeruginosa exported proteins using a consensus computational strategy combined with a laboratory-based PhoA fusion screen.Genome Res. 2005; 15 (15687295): 321-32910.1101/gr.3257305Crossref PubMed Scopus (98) Google Scholar). However, the fap system is evolutionarily younger than the curli system and only exists within a single phylum, the Proteobacteria (20Dueholm M.S. Otzen D. Nielsen P.H. Evolutionary insight into the functional amyloids of the pseudomonads.PLoS ONE. 2013; 8 (24116129): e7663010.1371/journal.pone.0076630Crossref PubMed Scopus (40) Google Scholar). Both amyloid proteins CsgA and FapC consist of multiple imperfect repeats. CsgA has five, each ca. 20 residues in length and folded as individual β-hairpins separated by a tight turn (4–5 residues), according to a computationally predicted structure (21Tian P. Boomsma W. Wang Y. Otzen D.E. Jensen M.H. Lindorff-Larsen K. Structure of a functional amyloid protein subunit computed using sequence variation.J. Am. Chem. Soc. 2015; 137 (25415595): 22-2510.1021/ja5093634Crossref PubMed Scopus (69) Google Scholar). In support of this, peptides corresponding to three of the individual repeats (repeats 1, 3, and 5) readily form amyloid on their own (22Wang X. Hammer N.D. Chapman M.R. The molecular basis of functional bacterial amyloid polymerization and nucleation.J. Biol. Chem. 2008; 283 (18508760): 21530-2153910.1074/jbc.M800466200Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). FapC has 3 longer repeats (ca. 35 residues long) that are also predicted to form β-hairpins (14Rouse S.L. Matthews S.J. Dueholm M.S. Ecology and biogenesis of functional amyloids in Pseudomonas.J. Mol. Biol. 2018; 430: 3685-369510.1016/j.jmb.2018.05.004Crossref PubMed Scopus (31) Google Scholar). However, in FapC the repeats are separated by linker regions of variable lengths; the second linker shows especially large variations in size, with lengths ranging from 39 (in the Pseudomonas sp. strain UK4 used in this study) to more than 250 residues for Pseudomonas putida F1 (13Dueholm M.S. Petersen S.V. Sønderkær M. Larsen P. Christiansen G. Hein K.L. Enghild J.J. Nielsen J.L. Nielsen K.L. Nielsen P.H. Otzen D.E. Functional amyloid in Pseudomonas.Mol. Microbiol. 2010; 77 (20572935): 1009-102010.1111/j.1365-2958.2010.07269.xCrossref PubMed Scopus (188) Google Scholar). For FapC, stepwise removal of these repeats has only relatively little effect on the rapidity of fibrillation but increases the ensuing fibrils' tendency to fragment during shaking, thereby forming new growing fibrils (23Rasmussen C.B. Christiansen G. Vad B.S. Lynggaard C. Enghild J.J. Andreasen M. Otzen D. Imperfect repeats in the functional amyloid protein FapC reduce the tendency to fragment during fibrillation.Protein Sci. 2019; 28 (30592554): 633-64210.1002/pro.3566Crossref PubMed Scopus (0) Google Scholar). Whereas that study focused on the mechanistic roles of the different repeats, we here address the question of how removal of these repeats affects the stability of the formed fibrils. Given that functional amyloids are largely made for structural reasons, it is their stability (thermodynamics) rather than the ease of their formation (kinetics) that is critical to their functionality. This aspect has not been addressed for functional amyloids. However, to answer this question thoroughly, we need a reliable assay to determine fibril stability. For globular monomeric proteins, stability is defined by the distribution between native and denatured states. Analogously, there is an equilibrium between the fibrillated and monomeric state; the higher the concentration of the monomeric species (corresponding to a critical aggregation concentration, or cac), the less stable the fibril. Consequently, the free energy of fibrillation can be expressed as –RT ln cac (24O'Nuallain B. Shivaprasad S. Kheterpal I. Wetzel R. Thermodynamics of A beta(1-40) amyloid fibril elongation.Biochemistry. 2005; 44 (16171385): 12709-1271810.1021/bi050927hCrossref PubMed Scopus (186) Google Scholar), and for relatively unstable amyloids formed by misfolded proteins involved in neurodegenerative diseases, this can be further analyzed by displacing the equilibrium toward the monomeric state by, e.g. addition of denaturants (25Baldwin A.J. Knowles T.P. Tartaglia G.G. Fitzpatrick A.W. Devlin G.L. Shammas S.L. Waudby C.A. Mossuto M.F. Meehan S. Gras S.L. Christodoulou J. Anthony-Cahill S.J. Barker P.D. Vendruscolo M. Dobson C.M. Metastability of native proteins and the phenomenon of amyloid formation.J. Am. Chem. Soc. 2011; 133 (21650202): 14160-1416310.1021/ja2017703Crossref PubMed Scopus (288) Google Scholar). This formalism has been used to evaluate the stability of the Aβ peptide, aided by the relatively high Aβ cac (24O'Nuallain B. Shivaprasad S. Kheterpal I. Wetzel R. Thermodynamics of A beta(1-40) amyloid fibril elongation.Biochemistry. 2005; 44 (16171385): 12709-1271810.1021/bi050927hCrossref PubMed Scopus (186) Google Scholar). However, fibrillation of functional amyloids such as FapC is very efficient and typically leads to impracticably low levels of monomer (13Dueholm M.S. Petersen S.V. Sønderkær M. Larsen P. Christiansen G. Hein K.L. Enghild J.J. Nielsen J.L. Nielsen K.L. Nielsen P.H. Otzen D.E. Functional amyloid in Pseudomonas.Mol. Microbiol. 2010; 77 (20572935): 1009-102010.1111/j.1365-2958.2010.07269.xCrossref PubMed Scopus (188) Google Scholar), even in the presence of high concentrations of denaturant (data not shown) or boiling SDS (13Dueholm M.S. Petersen S.V. Sønderkær M. Larsen P. Christiansen G. Hein K.L. Enghild J.J. Nielsen J.L. Nielsen K.L. Nielsen P.H. Otzen D.E. Functional amyloid in Pseudomonas.Mol. Microbiol. 2010; 77 (20572935): 1009-102010.1111/j.1365-2958.2010.07269.xCrossref PubMed Scopus (188) Google Scholar). Fortunately, other organic solvents are able to dissociate functional amyloids. For example, CsgA fibrils can be disassembled in a 1:1 mixture of hexafluoroisopropanol and TFA and subsequently reassembled in water or buffer (26Dorval Courchesne N.-M. Duraj-Thatte A. Tay P.K.R. Nguyen P.Q. Joshi N.S. Scalable production of genetically engineered nanofibrous macroscopic materials via filtration.ACS Biomater. Sci. Eng. 2017; 3: 733-74110.1021/acsbiomaterials.6b00437Crossref Scopus (48) Google Scholar), whereas fungal amyloids consisting of hydrophobins can be extracted with TFA and formic acid (FA) (27de Vries O.M.H. Fekkes M.P. Wösten H.A.B. Wessels J.G.H. Insoluble hydrophobin complexes in the walls of Schizophyllum commune and other filamentous fungi.Arch. Microbiol. 1993; 159: 330-33510.1007/BF00290915Crossref Scopus (175) Google Scholar). Overall, the simplest approach is to use the high concentrations (typically >80%) of FA that have been shown to solubilize functional amyloids. This phenomenon even serves as an operational basis to identify functional amyloid in complex mixtures (28Danielsen H.N. Hansen S.H. Herbst F.A. Kjeldal H. Stensballe A. Nielsen P.H. Dueholm M.S. Direct identification of functional amyloid proteins by label-free quantitative mass spectrometry.Biomolecules. 2017; 7: 5810.3390/biom7030058Crossref Scopus (9) Google Scholar). Therefore, the extent to which a protein fibril is dissolved by a progressive increase in FA may provide a measure of the fibril's stability. To investigate this systematically, however, we need a more general analysis of the denaturation potency of FA. To the best of our knowledge, such an analysis has not been performed yet, and here we provide it as part of our analysis of the stability of functional amyloid. Formic acid, HC(O)OH, is the simplest carboxylic acid. As a protein solvent, FA is superior to most common organic solvents (e.g. glycerol, DMSO, or TFA), solubilizing the protein polypeptide chain through protonation, destabilization of hydrogen bonds, and hydrophobic residue interactions (29Zheng S. Doucette A.A. Preventing N- and O-formylation of proteins when incubated in concentrated formic acid.Proteomics. 2016; 16 (26840995): 1059-106810.1002/pmic.201500366Crossref PubMed Scopus (23) Google Scholar, 30Houen G. Bechgaard K. Bechgaard K. Songstad J. Leskelä M. Polamo M. Homsi M.N. Kuske F.K.H. Haugg M. Trabesinger-Rüf N. Weinhold E.G. The solubility of proteins in organic solvents.Acta Chem. Scand. 1996; 50: 68-7010.3891/acta.chem.scand.50-0068Crossref Scopus (25) Google Scholar). High concentrations (>70%, v/v) of FA solubilize large, fibrous protein complexes, such as collagen, wool keratin, and silk fibroin (31Um I.C. Kweon H.Y. Lee K.G. Park Y.H. The role of formic acid in solution stability and crystallization of silk protein polymer.Int. J. Biol. Macromol. 2003; 33 (14607365): 203-21310.1016/j.ijbiomac.2003.08.004Crossref PubMed Scopus (128) Google Scholar, 32Aluigi A. Zoccola M. Vineis C. Tonin C. Ferrero F. Canetti M. Study on the structure and properties of wool keratin regenerated from formic acid.Int. J. Biol. Macromol. 2007; 41 (17467791): 266-27310.1016/j.ijbiomac.2007.03.002Crossref PubMed Scopus (202) Google Scholar, 33Eyre D.R. Glimcher M.J. The dissolution of bovine and chicken bone collagens in concentrated formic acid.Calcif. Tissue Res. 1974; 15 (4844385): 125-13210.1007/BF02059050Crossref PubMed Scopus (7) Google Scholar). Interestingly, the FA-solubilized form of fibroin seems to be more compact than that when solubilized in water, although fibroin is in random coil conformation in both solutions (31Um I.C. Kweon H.Y. Lee K.G. Park Y.H. The role of formic acid in solution stability and crystallization of silk protein polymer.Int. J. Biol. Macromol. 2003; 33 (14607365): 203-21310.1016/j.ijbiomac.2003.08.004Crossref PubMed Scopus (128) Google Scholar). Solubilization does not involve chemical modification (31Um I.C. Kweon H.Y. Lee K.G. Park Y.H. The role of formic acid in solution stability and crystallization of silk protein polymer.Int. J. Biol. Macromol. 2003; 33 (14607365): 203-21310.1016/j.ijbiomac.2003.08.004Crossref PubMed Scopus (128) Google Scholar); FA can formylate Thr and Ser (O-formylation) as well as Lys (N-formylation), but this requires high concentrations (>80%, v/v) and extended time intervals (many hours) (29Zheng S. Doucette A.A. Preventing N- and O-formylation of proteins when incubated in concentrated formic acid.Proteomics. 2016; 16 (26840995): 1059-106810.1002/pmic.201500366Crossref PubMed Scopus (23) Google Scholar). The amide derivative of FA, formamide (FM), is also able to solubilize and unfold globular proteins, such as myoglobin, cytochrome c, and insulin (34Herskovits T.T. Behrens C.F. Siuta P.B. Pandolfelli E.R. Solvent denaturation of globular proteins: unfolding by the monoalkyl- and dialkyl-substituted formamides and ureas.Biochim. Biophys. Acta. 1977; 490 (189823): 192-19910.1016/0005-2795(77)90119-2Crossref PubMed Scopus (30) Google Scholar, 35Choi T.S. Lee J.W. Jin K.S. Kim H.I. Amyloid fibrillation of insulin under water-limited conditions.Biophys. J. 2014; 107 (25418175): 1939-194910.1016/j.bpj.2014.09.008Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). Although not an acid, it still interacts with proteins through hydrogen bond disruption and solubilization of hydrophobic surfaces (36Khabiri M. Minofar B. Brezovsky J. Damborsky J. Ettrich R. Interaction of organic solvents with protein structures at protein-solvent interface.J. Mol. Model. 2013; 19 (22760789): 4701-471110.1007/s00894-012-1507-zCrossref PubMed Scopus (26) Google Scholar). Thus, FM constitutes a protonation-free comparison to FA. Our objectives for this study were 2-fold: (1) to establish a quantitative basis for the use of FA to determine protein stability and (2) to employ this approach to determine the contribution of the individual repeats in FapC to fibril stability. To achieve goal 1, we investigate the impact of FA on the stability of proteins that unfold according to a simple two-state system, making it straightforward to assess the impact of FA on their stability. However, given FA's properties as an acid, the simple lowering of pH will, in itself, make a major contribution to FA's destabilizing properties. To extract the non-protonation-related denaturing properties of FA, we need to be able to subtract the contributions that a simple drop in pH would make to destabilization. This restricts our investigations to proteins that do not unfold at low pH unless other denaturing measures are involved, such as heat. Therefore, we have selected three acid-resistant proteins (hen egg white lysozyme [37Parker M.J. Spencer J. Clarke A.R. An integrated kinetic analysis of intermediates and transition states in protein folding reactions.J. Mol. Biol. 1995; 253 (7473751): 771-78610.1006/jmbi.1995.0590Crossref PubMed Scopus (185) Google Scholar, 38Venkataramani S. Truntzer J. Coleman D.R. Thermal stability of high concentration lysozyme across varying pH: a Fourier transform infrared study.J. Pharm. Bioallied. Sci. 2013; 5 (23833521): 148-15310.4103/0975-7406.111821Crossref PubMed Scopus (62) Google Scholar, 39Wijaya E.C. Separovic F. Drummond C.J. Greaves T.L. Activity and conformation of lysozyme in molecular solvents, protic ionic liquids (PILs) and salt-water systems.Phys. Chem. Chem. Phys. 2016; 18 (27722290): 25926-2593610.1039/c6cp03334bCrossref PubMed Google Scholar, 40Wijaya E.C. Separovic F. Drummond C.J. Greaves T.L. Stability and activity of lysozyme in stoichiometric and non-stoichiometric protic ionic liquid (PIL)-water systems.J. Chem. Phys. 2018; 148 (30307182): 19383810.1063/1.5010055Crossref PubMed Scopus (22) Google Scholar], S6 from Thermus thermophilus [41Otzen D.E. Kristensen O. Proctor M. Oliveberg M. Structural changes in the transition state of protein folding: alternative interpretations of curved chevron plots.Biochemistry. 1999; 38 (10350468): 6499-651110.1021/bi982819jCrossref PubMed Scopus (180) Google Scholar, 42Otzen D.E. Oliveberg M. Correspondence between anomalous m- and DeltaCp-values in protein folding.Protein Sci. 2004; 13 (15557266): 3253-326310.1110/ps.04991004Crossref PubMed Scopus (0) Google Scholar, 43Otzen D.E. Oliveberg M. Burst-phase expansion of native protein prior to global unfolding in SDS.J. Mol. Biol. 2002; 315 (11827490): 1231-124010.1006/jmbi.2001.5300Crossref PubMed Scopus (69) Google Scholar, 44Otzen D.E. Nesgaard L.W. Andersen K.K. Hansen J.H. Christiansen G. Doe H. Sehgal P. Aggregation of S6 in a quasi-native state by sub-micellar SDS.Biochim. Biophys. Acta. 2008; 1784 (18083130): 400-41410.1016/j.bbapap.2007.11.010Crossref PubMed Scopus (31) Google Scholar], and bovine ubiquitin [45Khorasanizadeh S. Peters I.D. Butt T.R. Roder H. Folding and stability of a tryptophan-containing mutant of ubiquitin.Biochemistry. 1993; 32 (8392867): 7054-706310.1021/bi00078a034Crossref PubMed Scopus (228) Google Scholar, 46Wintrode P.L. Makhatadze G.I. Privalov P.L. Thermodynamics of ubiquitin unfolding.Proteins. 1994; 18 (8202465): 246-25310.1002/prot.340180305Crossref PubMed Scopus (152) Google Scholar, 47Surana P. Das R. Observing a late folding intermediate of ubiquitin at atomic resolution by NMR.Protein Sci. 2016; 25 (27111887): 1438-145010.1002/pro.2940Crossref PubMed Scopus (3) Google Scholar, 48Chyan C.L. Lin F.C. Peng H. Yuan J.M. Chang C.H. Lin S.H. Yang G. Reversible mechanical unfolding of single ubiquitin molecules.Biophys. J. 2004; 87 (15361414): 3995-400610.1529/biophysj.104.042754Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 49Ibarra-Molero B. Loladze V.V. Makhatadze G.I. Sanchez-Ruiz J.M. Thermal versus guanidine-induced unfolding of ubiquitin. An analysis in terms of the contributions from charge-charge interactions to protein stability.Biochemistry. 1999; 38 (10387059): 8138-814910.1021/bi9905819Crossref PubMed Scopus (199) Google Scholar, 50Jourdan M. Searle M.S. Insights into the stability of native and partially folded states of ubiquitin: effects of cosolvents and denaturants on the thermodynamics of protein folding.Biochemistry. 2001; 40 (11513610): 10317-1032510.1021/bi010767jCrossref PubMed Scopus (36) Google Scholar, 51Milardi D. Arnesano F. Grasso G. Magri A. Tabbi G. Scintilla S. Natile G. Rizzarelli E. Ubiquitin stability and the Lys63-linked polyubiquitination site are compromised on copper binding.Angew. Chem. Int. Ed. Engl. 2007; 46 (17854105): 7993-799510.1002/anie.200701987Crossref PubMed Scopus (36) Google Scholar]) whose stabilities all have been extensively analyzed under a range of conditions. For all three proteins, we determine their stability in FA based on near-UV CD thermal scans over a range of FA concentrations. For each FA concentration, we carry out a thermal scan at the corresponding pH adjusted with HCl and subtract this effect from that of FA to quantitate the intrinsic contribution of FA to protein stability. Furthermore, we carry out these experiments using <30%, v/v, FA to minimize formylation (29Zheng S. Doucette A.A. Preventing N- and O-formylation of proteins when incubated in concentrated formic acid.Proteomics. 2016; 16 (26840995): 1059-106810.1002/pmic.201500366Crossref PubMed Scopus (23) Google Scholar). To achieve goal 2, we use the obtained relationship between FA concentration and its denaturation potency (m-values) to evaluate the FA depolymerization assays of eight different FapC constructs (Fig. S1) and thereby quantify the aggregative effect of individual repeats of FapC. To quantitate the denaturing potency of formic acid (FA), we used near-UV CD to carry out thermal scans of three model proteins in different concentrations of FA. The proteins are the 129-residue hen egg white lysozyme, the 101-residue ribosomal protein S6 from Thermus thermophiles, and the 76-residue bovine ubiquitin (Ubi). These proteins were selected for a number of reasons. First, they are well-characterized small proteins in a range of sizes (from 8.6 kDa through 12 kDa to 16.2 kDa) that have previously been used as model systems for fundamental studies in protein stability and folding (37Parker M.J. Spencer J. Clarke A.R. An integrated kinetic analysis of intermediates and transition states in protein folding reactions.J. Mol. Biol. 1995; 253 (7473751): 771-78610.1006/jmbi.1995.0590Crossref PubMed Scopus (185) Google Scholar, 38Venkataramani S. Truntzer J. Coleman D.R. Thermal stability of high concentration lysozyme across" @default.
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• W3043970664 title "Quantitating denaturation by formic acid: imperfect repeats are essential to the stability of the functional amyloid protein FapC" @default.
• W3043970664 cites W1491886680 @default.
• W3043970664 cites W1606043635 @default.
• W3043970664 cites W1847069914 @default.
• W3043970664 cites W1935984619 @default.
• W3043970664 cites W1964517967 @default.
• W3043970664 cites W1968201804 @default.
• W3043970664 cites W1977820570 @default.
• W3043970664 cites W1981625739 @default.
• W3043970664 cites W1983439313 @default.
• W3043970664 cites W1989451911 @default.
• W3043970664 cites W1995281770 @default.
• W3043970664 cites W2003319013 @default.
• W3043970664 cites W2007760903 @default.
• W3043970664 cites W2010230246 @default.
• W3043970664 cites W2012124434 @default.
• W3043970664 cites W2015461118 @default.
• W3043970664 cites W2020563037 @default.
• W3043970664 cites W2021785060 @default.
• W3043970664 cites W2026944026 @default.
• W3043970664 cites W2028246897 @default.
• W3043970664 cites W2046690916 @default.
• W3043970664 cites W2063827082 @default.
• W3043970664 cites W2066859686 @default.
• W3043970664 cites W2066933443 @default.
• W3043970664 cites W2072792187 @default.
• W3043970664 cites W2075734803 @default.
• W3043970664 cites W2077884408 @default.
• W3043970664 cites W2080196868 @default.
• W3043970664 cites W2080880014 @default.
• W3043970664 cites W2082314807 @default.
• W3043970664 cites W2085401124 @default.
• W3043970664 cites W2085467932 @default.
• W3043970664 cites W2091086159 @default.
• W3043970664 cites W2094135937 @default.
• W3043970664 cites W2100877559 @default.
• W3043970664 cites W2103114542 @default.
• W3043970664 cites W2117747873 @default.
• W3043970664 cites W2119376894 @default.
• W3043970664 cites W2122680286 @default.
• W3043970664 cites W2123736620 @default.
• W3043970664 cites W2129507901 @default.
• W3043970664 cites W2130607734 @default.
• W3043970664 cites W2133067061 @default.
• W3043970664 cites W2139254511 @default.
• W3043970664 cites W2145626269 @default.
• W3043970664 cites W2157529471 @default.
• W3043970664 cites W2165028383 @default.
• W3043970664 cites W2165064655 @default.
• W3043970664 cites W2165548028 @default.
• W3043970664 cites W2171132114 @default.
• W3043970664 cites W2171242122 @default.
• W3043970664 cites W2262864248 @default.
• W3043970664 cites W2321593295 @default.
• W3043970664 cites W2330959820 @default.
• W3043970664 cites W2333694243 @default.
• W3043970664 cites W2343158053 @default.
• W3043970664 cites W2518204164 @default.
• W3043970664 cites W2559466007 @default.
• W3043970664 cites W2729573548 @default.
• W3043970664 cites W2742302497 @default.
• W3043970664 cites W2743955849 @default.
• W3043970664 cites W2793188028 @default.
• W3043970664 cites W2803584042 @default.
• W3043970664 cites W2811383464 @default.
• W3043970664 cites W2908175056 @default.
• W3043970664 cites W2916996208 @default.
• W3043970664 cites W2967201176 @default.
• W3043970664 cites W2973147842 @default.
• W3043970664 cites W2981733277 @default.
• W3043970664 cites W4232223138 @default.
• W3043970664 cites W4255258217 @default.
• W3043970664 cites W1970496660 @default.
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