Donor strand sequence, rather than donor strand orientation, determines the stability and non-equilibrium folding of the type 1 pilus subunit FimA

Abstract

FimA is the main structural subunit of adhesive type 1 pili from uropathogenic Escherichia coli strains. Up to 3000 copies of FimA assemble to the helical pilus rod through a mechanism termed donor strand complementation, in which the incomplete immunoglobulin-like fold of each FimA subunit is complemented by the N-terminal extension (Nte) of the next subunit. The Nte of FimA, which exhibits a pseudo-palindromic sequence, is inserted in an antiparallel orientation relative to the last β-strand of the preceding subunit in the pilus. The resulting subunit-subunit interactions are extraordinarily stable against dissociation and unfolding. Alternatively, FimA can fold to a self-complemented monomer with anti-apoptotic activity, in which the Nte inserts intramolecularly into the FimA core in the opposite, parallel orientation. The FimA monomers, however, show dramatically lower thermodynamic stability compared with FimA subunits in the assembled pilus. Using self-complemented FimA variants with reversed, pseudo-palindromic extensions, we demonstrate that the high stability of FimA polymers is primarily caused by the specific interactions between the side chains of the Nte residues and the FimA core and not by the antiparallel orientation of the donor strand alone. In addition, we demonstrate that nonequilibrium two-state folding, a hallmark of FimA with the Nte inserted in the pilus rod-like, antiparallel orientation, only depends on the identity of the inserted Nte side chains and not on Nte orientation. FimA is the main structural subunit of adhesive type 1 pili from uropathogenic Escherichia coli strains. Up to 3000 copies of FimA assemble to the helical pilus rod through a mechanism termed donor strand complementation, in which the incomplete immunoglobulin-like fold of each FimA subunit is complemented by the N-terminal extension (Nte) of the next subunit. The Nte of FimA, which exhibits a pseudo-palindromic sequence, is inserted in an antiparallel orientation relative to the last β-strand of the preceding subunit in the pilus. The resulting subunit-subunit interactions are extraordinarily stable against dissociation and unfolding. Alternatively, FimA can fold to a self-complemented monomer with anti-apoptotic activity, in which the Nte inserts intramolecularly into the FimA core in the opposite, parallel orientation. The FimA monomers, however, show dramatically lower thermodynamic stability compared with FimA subunits in the assembled pilus. Using self-complemented FimA variants with reversed, pseudo-palindromic extensions, we demonstrate that the high stability of FimA polymers is primarily caused by the specific interactions between the side chains of the Nte residues and the FimA core and not by the antiparallel orientation of the donor strand alone. In addition, we demonstrate that nonequilibrium two-state folding, a hallmark of FimA with the Nte inserted in the pilus rod-like, antiparallel orientation, only depends on the identity of the inserted Nte side chains and not on Nte orientation. Type 1 pili of uropathogenic Escherichia coli (UPEC) strains are filamentous surface protein complexes that mediate pathogen attachment to urinary epithelium cells by binding high-mannose-type glycans of surface glycoproteins via the lectin FimH at their distal tip (1Ronald A. The etiology of urinary tract infection: traditional and emerging pathogens.Dis. Mon. 2003; 49 (12601338): 71-8210.1067/mda.2003.8Crossref PubMed Scopus (0) Google Scholar, 2Sauer M.M. Jakob R.P. Luber T. Canonica F. Navarra G. Ernst B. Unverzagt C. Maier T. Glockshuber R. Binding of the bacterial adhesin FimH to its natural, multivalent high-mannose type glycan targets.J. Am. Chem. Soc. 2019; 141 (30543411): 936-94410.1021/jacs.8b10736Crossref PubMed Scopus (24) Google Scholar, 3Hospenthal M.K. Waksman G. The remarkable biomechanical properties of the type 1 chaperone-usher pilus: a structural and molecular perspective.Microbiol. Spectr. 2019; 7 (30681068)10.1128/microbiolspec.psib-0010-2018PubMed Google Scholar, 4Flores-Mireles A.L. Walker J.N. Caparon M. Hultgren S.J. Urinary tract infections: epidemiology, mechanisms of infection and treatment options.Nat. Rev. Microbiol. 2015; 13 (25853778): 269-28410.1038/nrmicro3432Crossref PubMed Scopus (1110) Google Scholar). The type 1 pilus is composed of the helical pilus rod formed by up to 3000 copies of the main pilus subunit FimA and a linear tip fibrillum formed by the minor subunits FimF and FimG and the adhesin FimH. Pilus assembly in vivo proceeds via the chaperone-usher pathway (5Thanassi D.G. Saulino E.T. Hultgren S.J. The chaperone/usher pathway: a major terminal branch of the general secretory pathway.Curr. Opin. Microbiol. 1998; 1 (10066482): 223-23110.1016/S1369-5274(98)80015-5Crossref PubMed Scopus (151) Google Scholar), in which the periplasmic chaperone FimC catalyzes the folding of the pilus subunits as soon as their invariant, structural disulfide bond is formed by disulfide exchange with the periplasmic dithiol oxidase DsbA (6Crespo M.D. Puorger C. Schärer M.A. Eidam O. Grütter M.G. Capitani G. Glockshuber R. Quality control of disulfide bond formation in pilus subunits by the chaperone FimC.Nat. Chem. Biol. 2012; 8 (22772153): 707-71310.1038/nchembio.1019Crossref PubMed Scopus (40) Google Scholar). FimC then delivers the folded subunits to the assembly platform (usher) FimD in the outer membrane (7Vetsch M. Erilov D. Molière N. Nishiyama M. Ignatov O. Glockshuber R. Mechanism of fibre assembly through the chaperone-usher pathway.EMBO Rep. 2006; 7 (16767077): 734-73810.1038/sj.embor.7400722Crossref PubMed Scopus (48) Google Scholar), where FimD catalyzes subunit assembly and mediates subunit translocation across the outer membrane (Fig. 1A) (8Geibel S. Procko E. Hultgren S.J. Baker D. Waksman G. Structural and energetic basis of folded-protein transport by the FimD usher.Nature. 2013; 496 (23579681): 243-24610.1038/nature12007Crossref PubMed Scopus (69) Google Scholar, 9Nishiyama M. Horst R. Eidam O. Herrmann T. Ignatov O. Vetsch M. Bettendorff P. Jelesarov I. Grütter M.G. Wüthrich K. Glockshuber R. Capitani G. Structural basis of chaperone-subunit complex recognition by the type 1 pilus assembly platform FimD.EMBO J. 2005; 24 (15920478): 2075-208610.1038/sj.emboj.7600693Crossref PubMed Scopus (98) Google Scholar). The subunits in the assembled pilus form highly stable interactions that confer infinite stability against spontaneous dissociation to the pilus (10Żyła D.S. Prota A.E. Capitani G. Glockshuber R. Alternative folding to a monomer or homopolymer is a common feature of the type 1 pilus subunit FimA from enteroinvasive bacteria.J. Biol. Chem. 2019; 294 (31126987): 10553-1056310.1074/jbc.RA119.008610Abstract Full Text Full Text PDF PubMed Scopus (2) Google Scholar, 11Puorger C. Eidam O. Capitani G. Erilov D. Grütter M.G. Glockshuber R. Infinite kinetic stability against dissociation of supramolecular protein complexes through donor strand complementation.Structure. 2008; 16 (18400183): 631-64210.1016/j.str.2008.01.013Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). The common mechanism of the subunit-subunit interactions is termed donor strand complementation, in which the incomplete, immunoglobulin-like (pilin) fold of each subunit, lacking the C-terminal β-strand, is completed by insertion of the N-terminal ∼20-residue extension of the following subunit that provides the missing β-strand (12Barnhart M.M. S. Waksman G. C. Hultgren S.J. the missing folding of S. A. PubMed Scopus Google Scholar, M.M. Hultgren S.J. interactions donor strand exchange bacterial pilus 2003; PubMed Scopus (53) Google Scholar). the N-terminal extension termed donor of the next subunit inserts in an antiparallel orientation relative to the C-terminal β-strand of the fold into a in the The which Nte side chains (12Barnhart M.M. S. Waksman G. C. Hultgren S.J. the missing folding of S. A. PubMed Scopus Google Scholar, C. Vetsch M. G. Glockshuber R. folding and stability of FimA, the main structural subunit of type 1 pili from uropathogenic Escherichia coli Biol. PubMed Scopus Google Scholar, A. J. S. F. D. E. R. and folding of E. coli type 1 pilus PubMed Scopus Google Scholar). The main pilus subunit FimA from type 1 enteroinvasive coli from pilus subunits in that folding can assemble to the pilus rod E. P. P. Glockshuber R. M. N. P. the molecular of Escherichia coli type 1 Biol. PubMed Scopus Google via the antiparallel donor strand mechanism or can fold to self-complemented in which the Nte inserts intramolecularly into the FimA fold in the opposite, parallel orientation (10Żyła D.S. Prota A.E. Capitani G. Glockshuber R. Alternative folding to a monomer or homopolymer is a common feature of the type 1 pilus subunit FimA from enteroinvasive bacteria.J. Biol. Chem. 2019; 294 (31126987): 10553-1056310.1074/jbc.RA119.008610Abstract Full Text Full Text PDF PubMed Scopus (2) Google Scholar, Puorger C. Glockshuber R. G. donor strand in the E. coli type 1 pilus subunit FimA the of FimA as of pilus assembly that Biol. PubMed Scopus Google Scholar). folding to a pseudo-palindromic in the Nte of FimA in E. coli with in the of the where is a and is a The of E. coli FimA that the residues and which the in the FimA monomer and the FimA the of the inserted Nte The is that can only a the between the inserted Nte and the and of the FimA fold (Fig. (10Żyła D.S. Prota A.E. Capitani G. Glockshuber R. Alternative folding to a monomer or homopolymer is a common feature of the type 1 pilus subunit FimA from enteroinvasive bacteria.J. Biol. Chem. 2019; 294 (31126987): 10553-1056310.1074/jbc.RA119.008610Abstract Full Text Full Text PDF PubMed Scopus (2) Google Scholar, C. Vetsch M. G. Glockshuber R. folding and stability of FimA, the main structural subunit of type 1 pili from uropathogenic Escherichia coli Biol. PubMed Scopus Google Scholar, Puorger C. Glockshuber R. G. donor strand in the E. coli type 1 pilus subunit FimA the of FimA as of pilus assembly that Biol. PubMed Scopus Google Scholar). The of the FimA into the E. coli to the assembly chaperone fold to and are as complexes to the assembly platform a of the FimA to fold of FimC to FimA as of of urinary epithelium cells type 1 mechanism strains to treatment urinary tract the cells form of the pilus protein FimA the complex at to Full Text Full Text PDF PubMed Scopus Google Scholar). The of Nte insertion into the FimA fold folding and thermodynamic stability of that the self-complemented E. coli FimA monomer exhibits only thermodynamic of the E. coli FimA monomer 1 of and of folding of only at and (10Żyła D.S. Prota A.E. Capitani G. Glockshuber R. Alternative folding to a monomer or homopolymer is a common feature of the type 1 pilus subunit FimA from enteroinvasive bacteria.J. Biol. Chem. 2019; 294 (31126987): 10553-1056310.1074/jbc.RA119.008610Abstract Full Text Full Text PDF PubMed Scopus (2) Google Scholar, C. Vetsch M. G. Glockshuber R. folding and stability of FimA, the main structural subunit of type 1 pili from uropathogenic Escherichia coli Biol. PubMed Scopus Google Scholar). In a self-complemented FimA the of FimA in the assembled pilus to with a of folding of (10Żyła D.S. Prota A.E. Capitani G. Glockshuber R. Alternative folding to a monomer or homopolymer is a common feature of the type 1 pilus subunit FimA from enteroinvasive bacteria.J. Biol. Chem. 2019; 294 (31126987): 10553-1056310.1074/jbc.RA119.008610Abstract Full Text Full Text PDF PubMed Scopus (2) Google Scholar, C. Vetsch M. G. Glockshuber R. folding and stability of FimA, the main structural subunit of type 1 pili from uropathogenic Escherichia coli Biol. PubMed Scopus Google Scholar). In a of the Nte to the FimA via a C-terminal Nte inserts in the antiparallel orientation into the FimA the Nte at the (Fig. and In addition, high and that the folding at the of 1 only at only 1 of the and of to with the two-state of that the stability of FimA in the of the assembled pilus is on its infinite stability against as the of spontaneous at and to in the of (10Żyła D.S. Prota A.E. Capitani G. Glockshuber R. Alternative folding to a monomer or homopolymer is a common feature of the type 1 pilus subunit FimA from enteroinvasive bacteria.J. Biol. Chem. 2019; 294 (31126987): 10553-1056310.1074/jbc.RA119.008610Abstract Full Text Full Text PDF PubMed Scopus (2) Google Scholar). In the we the of the stability of FimA with the Nte inserted in the antiparallel orientation in in in the pilus primarily from antiparallel β-strand insertion or from specific interactions between the Nte side chains of the Nte in the binding we compared the stability of FimA and with that of FimA FimA and FimA in which the Nte (Fig. and and In FimA the of the FimA residues relative to In the FimA the Nte to the via a and the Nte demonstrate that the of FimA in the of the pilus stability against and folding are by the identity of the Nte side chains in the of the FimA FimA variants in the E. coli the control of the the FimA to the in the variants to form the structural disulfide bond and as FimA and FimA and FimA by (10Żyła D.S. Prota A.E. Capitani G. Glockshuber R. Alternative folding to a monomer or homopolymer is a common feature of the type 1 pilus subunit FimA from enteroinvasive bacteria.J. Biol. Chem. 2019; 294 (31126987): 10553-1056310.1074/jbc.RA119.008610Abstract Full Text Full Text PDF PubMed Scopus (2) Google Scholar). the formation of the structural disulfide bond by in the of the variants by of the through and by on in which at the to the folded FimA The of FimA variants of bacterial that the N-terminal in FimA In a to the stability of FimA relative to that of FimA and of relative to that of FimA we of at via the of the at of the Nte in FimA caused a in the FimA with an in the by from 49 to (Fig. the of the donor strand to the of FimA stability with an of the of the protein to 49 in the FimA the the FimA (Fig. the that the Nte residues into the of the fold of FimA are the stability of donor FimA and that the antiparallel donor strand orientation is not to the stability of FimA subunits in the assembled pilus. we and at and of FimA and FimA that FimA to the FimA its folding 1 FimA, FimA to only with a of folding of is to the stability of FimA the is with the against of FimA and FimA (Fig. and kinetic folding of the FimA with donor strand side chains into the FimA fold as in FimA that a folding 1 from that a folding 1 that a folding 1 from D.S. Prota A.E. Capitani G. Glockshuber R. Alternative folding to a monomer or homopolymer is a common feature of the type 1 pilus subunit FimA from enteroinvasive bacteria.J. Biol. Chem. 2019; 294 (31126987): 10553-1056310.1074/jbc.RA119.008610Abstract Full Text Full Text PDF PubMed Scopus (2) Google in a and kinetic folding of the FimA with donor strand side chains into the FimA fold as in interactions in the assembled pilus to between and that not a folding 8 from that not a folding 8 to between and that not a folding 8 from D.S. Prota A.E. Capitani G. Glockshuber R. Alternative folding to a monomer or homopolymer is a common feature of the type 1 pilus subunit FimA from enteroinvasive bacteria.J. Biol. Chem. 2019; 294 (31126987): 10553-1056310.1074/jbc.RA119.008610Abstract Full Text Full Text PDF PubMed Scopus (2) Google in a the 1 and 8 of of FimA from FimA and FimA 8 the folding of FimA not only at high and only at (Fig. The of and to with a two-state with a high and the of the of on the from the nonequilibrium in which the of folding and the of folding and at of FimA The of that FimA is stable the FimA FimA and FimA show the donor strand orientation and only in their Nte the demonstrate that only the Nte residues with the of the FimA fold the of FimA subunits in the assembled pilus thermodynamic stability and kinetic stability against and dissociation that the E. coli FimA monomer and and with of (10Żyła D.S. Prota A.E. Capitani G. Glockshuber R. Alternative folding to a monomer or homopolymer is a common feature of the type 1 pilus subunit FimA from enteroinvasive bacteria.J. Biol. Chem. 2019; 294 (31126987): 10553-1056310.1074/jbc.RA119.008610Abstract Full Text Full Text PDF PubMed Scopus (2) Google Scholar, C. Vetsch M. G. Glockshuber R. folding and stability of FimA, the main structural subunit of type 1 pili from uropathogenic Escherichia coli Biol. PubMed Scopus Google Scholar), that residues that form the Nte not to the stability of the of that the FimA variants FimA and FimA fold with we the at and of FimA FimA FimA, and the with FimA variants with the between and that the of folding are the FimA variants (Fig. the and FimA which not folding at the of folding in (Fig. and a of with from their nonequilibrium folding at the (Fig. The are with a in which the of FimA folding is only formed by the residues of the FimA fold C. Vetsch M. G. Glockshuber R. folding and stability of FimA, the main structural subunit of type 1 pili from uropathogenic Escherichia coli Biol. PubMed Scopus Google Scholar). is the in FimA variants In addition, the that the orientation of the β-strand that the FimA fold its the FimA folding variants fold with the their thermodynamic are dramatically the of the and C-terminal in the FimA and FimA inserted as into the FimA we the of variants at and by molecular the of FimA as a (Fig. and of the of FimA and FimA and of and FimA that the of the and C-terminal by the immunoglobulin-like FimA fold of and the in the donor strand the of β-strand insertion of the and by the residues the and of the FimA fold (Fig. In addition, the of FimA variants that the and can or side of the orientation of the donor the in thermodynamic stability between FimA and FimA and between and FimA we not specific structural that stability The only in the stable and FimA that their donor a of residues compared with the donor in the stable variants FimA and FimA (Fig. the a of the donor strand of in the (Fig. only donor residues of in the (Fig. and demonstrate that FimA variants fold to the two-state and that in thermodynamic stability from in the of unfolding. The two-state folding that interactions the are unfolding. structural the highly and on and the of FimA and FimA in the the in of of in the the in a the structural the of FimA variants with thermodynamic in which the are by residues the donor the orientation. of to highly high and their orientation, the donor the pseudo-palindromic and the of the side chains in the the (Fig. the structural that the identity of the side chains in the and not the orientation of the donor strand that inserts into the FimA is the main of the stability and folding of The assembly of FimA to that form the helical of the pilus rod is in vivo by the periplasmic folding FimC (6Crespo M.D. Puorger C. Schärer M.A. Eidam O. Grütter M.G. Capitani G. Glockshuber R. Quality control of disulfide bond formation in pilus subunits by the chaperone FimC.Nat. Chem. Biol. 2012; 8 (22772153): 707-71310.1038/nchembio.1019Crossref PubMed Scopus (40) Google Scholar, C. Vetsch M. G. Glockshuber R. folding and stability of FimA, the main structural subunit of type 1 pili from uropathogenic Escherichia coli Biol. PubMed Scopus Google and the assembly platform FimD in the outer membrane M. T. Glockshuber R. of pilus assembly a bacterial outer membrane 2008; PubMed Scopus Google Scholar). FimA can assemble to pilus in in the of (Fig. M.K. D. A. C. J. Glockshuber R. Waksman G. The of the type 1 chaperone-usher pilus Full Text Full Text PDF PubMed Scopus Google Scholar). however, FimA assemble to at with a of at FimA monomer of (Fig. and The assembly is the that folded FimA are and to can with the Nte of FimA (Fig. the in spontaneous FimA a between a folded and an FimA the FimA core fold to an C. Vetsch M. G. Glockshuber R. folding and stability of FimA, the main structural subunit of type 1 pili from uropathogenic Escherichia coli Biol. PubMed Scopus Google Scholar). The following assembly the of on the The subunit are to (10Żyła D.S. Prota A.E. Capitani G. Glockshuber R. Alternative folding to a monomer or homopolymer is a common feature of the type 1 pilus subunit FimA from enteroinvasive bacteria.J. Biol. Chem. 2019; 294 (31126987): 10553-1056310.1074/jbc.RA119.008610Abstract Full Text Full Text PDF PubMed Scopus (2) Google Scholar, C. Vetsch M. G. Glockshuber R. folding and stability of FimA, the main structural subunit of type 1 pili from uropathogenic Escherichia coli Biol. PubMed Scopus Google Scholar). The thermodynamic stability of FimA spontaneous FimA assembly a of the is In that of the of the FimA monomer or spontaneous we the of homopolymer formation of the FimA which a of folding of and an of the spontaneous of FimA 7 where of FimA assembled to (Fig. and its stability against the of FimA to form the lower stability of FimA antiparallel donor strand insertion and of FimA not stable the FimA in to FimA (Fig. C. Eidam O. Capitani G. Erilov D. Grütter M.G. Glockshuber R. Infinite kinetic stability against dissociation of supramolecular protein complexes through donor strand complementation.Structure. 2008; 16 (18400183): 631-64210.1016/j.str.2008.01.013Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar), the formation of subunit-subunit not in the of FimA and the formation of stable pilus in The bacterial type 1 pilus subunit FimA from to its remarkable to fold into with The of the FimA assemble to the highly stable helical of the pilus in which the FimA subunits via antiparallel donor strand M.K. D. A. C. J. Glockshuber R. Waksman G. The of the type 1 chaperone-usher pilus Full Text Full Text PDF PubMed Scopus Google Scholar, Hultgren S.J. pili in and PubMed Scopus Google Scholar). a of the FimA to self-complemented that of urinary cells type 1 of form of the pilus protein FimA the complex at to Full Text Full Text PDF PubMed Scopus Google Scholar). The folding is the pseudo-palindromic of the β-strand formed by the N-terminal extension of FimA (10Żyła D.S. Prota A.E. Capitani G. Glockshuber R. Alternative folding to a monomer or homopolymer is a common feature of the type 1 pilus subunit FimA from enteroinvasive bacteria.J. Biol. Chem. 2019; 294 (31126987): 10553-1056310.1074/jbc.RA119.008610Abstract Full Text Full Text PDF PubMed Scopus (2) Google Scholar, C. Vetsch M. G. Glockshuber R. folding and stability of FimA, the main structural subunit of type 1 pili from uropathogenic Escherichia coli Biol. PubMed Scopus Google Scholar, Puorger C. Glockshuber R. G. donor strand in the E. coli type 1 pilus subunit FimA the of FimA as of pilus assembly that Biol. PubMed Scopus Google Scholar). In the we the of pseudo-palindromic sequence, which can the immunoglobulin-like of FimA by into the FimA fold as a β-strand in folding and stability of that the in thermodynamic stability between the FimA is by the identity of the residues into the donor strand binding and as C. Vetsch M. G. Glockshuber R. folding and stability of FimA, the main structural subunit of type 1 pili from uropathogenic Escherichia coli Biol. PubMed Scopus Google Scholar, Puorger C. Glockshuber R. G. donor strand in the E. coli type 1 pilus subunit FimA the of FimA as of pilus assembly that Biol. PubMed Scopus Google Scholar), by the orientation or the of the inserted donor the FimA fold the reversed, donor strand in FimA FimA and FimA In addition, FimA variants the properties of the and the FimA which donor strand side chains in the properties are thermodynamic stability and two-state folding in the of the and stability with two-state folding in the of the The residues in the and are in FimA and and the of the donor strand side and which can a or a side to the main of FimA folding and in E. coli control of FimA and (10Żyła D.S. Prota A.E. Capitani G. Glockshuber R. Alternative folding to a monomer or homopolymer is a common feature of the type 1 pilus subunit FimA from enteroinvasive bacteria.J. Biol. Chem. 2019; 294 (31126987): 10553-1056310.1074/jbc.RA119.008610Abstract Full Text Full Text PDF PubMed Scopus (2) Google Scholar, C. Vetsch M. G. Glockshuber R. folding and stability of FimA, the main structural subunit of type 1 pili from uropathogenic Escherichia coli Biol. PubMed Scopus Google Scholar). FimA and FimA from with molecular The FimA variants FimA and FimA in and as FimA and (10Żyła D.S. Prota A.E. Capitani G. Glockshuber R. Alternative folding to a monomer or homopolymer is a common feature of the type 1 pilus subunit FimA from enteroinvasive bacteria.J. Biol. Chem. 2019; 294 (31126987): 10553-1056310.1074/jbc.RA119.008610Abstract Full Text Full Text PDF PubMed Scopus (2) Google Scholar). in E. coli cells the at in at by the of an and by of the in an protein and at The with of and of The at and to and membrane The with and to and and then at in of by and and by exchange on a with The protein then to with and and at and formation of the structural disulfide bond in the FimA The to a protein of by membrane and by against and 1 The then to by and to a with the the and to 1 of protein in and at by FimA that the N-terminal The of protein of bacterial each FimA of FimA variants via their specific at the N-terminal extension of FimA and variants the at of in to a of and at to unfolding. by to protein with at by the in the at a The in the and via the The of PubMed Scopus Google Scholar). with and with to where is the is the at is the at the of the is the of at and is the in to The of the at from to at the of 1 with to a two-state of 2003; 13 PubMed Scopus Google where is the and are the of and folded protein at and are the of the of and is the is the of is the and is the in K. The then and the of against only the as a of we not of unfolding. of folded in or in and and at to in and at The at of each and The via their The of PubMed Scopus Google Scholar). The against FimA folding and 1 of The to the two-state of folding to where is the and are the of and protein at and are the of the of and is the of folding at and is the of of The folding of FimA not at and to the of an two-state a of as D. Puorger C. Glockshuber R. of protein folding Am. Chem. Soc. PubMed Scopus Google where and are the of folding and at and are the linear of and on is the and is the of The is and the of of a two-state folding and and the of and on of the 7 where is the and and are the of the folded and protein at and and are the of and the to The the FimA folding (Fig. from the in the between and of folded in the of folded and the the of the of against and to with The of the FimA in from where is the of folding at and is the of folding which and in from to up of FimA or FimA in with a up at and of FimA at in with the of as of FimA at in of as and in FimA and FimA at at the with a of 1 and a with Biol. PubMed Scopus Google and at FimA and in FimA by molecular FimA FimA and FimA in from the G. N. R. a Biol. PubMed Scopus Google Scholar). and in P. B. K. and of Biol. PubMed Scopus Google and The are FimA and FimA and on and on only in the a to the between the in the of FimA against the in the between donor and the FimA core with the A. R. R. a to and at the of PubMed Scopus Google Scholar). donor strand as and FimA or FimA in to a protein of and at by on a with the of the assembly or 1 The of the at 1 the of from the and with at on at in to the with the following and the S. with the of the and B. with up the and from the with M. S. and P. with uropathogenic Escherichia coli N-terminal extension C-terminal extension

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