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• W1968875837 abstract "The redesign of biological nanopores is focused on bacterial outer membrane proteins and pore-forming toxins, because their robust β-barrel structure makes them the best choice for developing stochastic biosensing elements. Using membrane protein engineering and single-channel electrical recordings, we explored the ferric hydroxamate uptake component A (FhuA), a monomeric 22-stranded β-barrel protein from the outer membrane of Escherichia coli. FhuA has a luminal cross-section of 3.1 × 4.4 nm and is filled by a globular N-terminal cork domain. Various redesigned FhuA proteins were investigated, including single, double, and multiple deletions of the large extracellular loops and the cork domain. We identified four large extracellular loops that partially occlude the lumen when the cork domain is removed. The newly engineered protein, FhuAΔC/Δ4L, was the result of a removal of almost one-third of the total number of amino acids of the wild-type FhuA (WT-FhuA) protein. This extensive protein engineering encompassed the entire cork domain and four extracellular loops. Remarkably, FhuAΔC/Δ4L forms a functional open pore in planar lipid bilayers, with a measured unitary conductance of ∼4.8 nanosiemens, which is much greater than the values recorded previously with other engineered FhuA protein channels. There are numerous advantages and prospects of using such an engineered outer membrane protein not only in fundamental studies of membrane protein folding and design, and the mechanisms of ion conductance and gating, but also in more applicative areas of stochastic single-molecule sensing of proteins and nucleic acids. The redesign of biological nanopores is focused on bacterial outer membrane proteins and pore-forming toxins, because their robust β-barrel structure makes them the best choice for developing stochastic biosensing elements. Using membrane protein engineering and single-channel electrical recordings, we explored the ferric hydroxamate uptake component A (FhuA), a monomeric 22-stranded β-barrel protein from the outer membrane of Escherichia coli. FhuA has a luminal cross-section of 3.1 × 4.4 nm and is filled by a globular N-terminal cork domain. Various redesigned FhuA proteins were investigated, including single, double, and multiple deletions of the large extracellular loops and the cork domain. We identified four large extracellular loops that partially occlude the lumen when the cork domain is removed. The newly engineered protein, FhuAΔC/Δ4L, was the result of a removal of almost one-third of the total number of amino acids of the wild-type FhuA (WT-FhuA) protein. This extensive protein engineering encompassed the entire cork domain and four extracellular loops. Remarkably, FhuAΔC/Δ4L forms a functional open pore in planar lipid bilayers, with a measured unitary conductance of ∼4.8 nanosiemens, which is much greater than the values recorded previously with other engineered FhuA protein channels. There are numerous advantages and prospects of using such an engineered outer membrane protein not only in fundamental studies of membrane protein folding and design, and the mechanisms of ion conductance and gating, but also in more applicative areas of stochastic single-molecule sensing of proteins and nucleic acids. One critical prerequisite for developing a sensitive stochastic sensing element is a robust protein scaffold (1Bayley H. Cremer P.S. Nature. 2001; 413: 226-230Crossref PubMed Scopus (965) Google Scholar, 2Movileanu L. Trends Biotechnol. 2009; 27: 333-341Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar, 3Howorka S. Siwy Z. Chem. Soc. Rev. 2009; 38: 2360-2384Crossref PubMed Scopus (941) Google Scholar). Recent studies in structural biology have revealed that β-barrel membrane proteins fulfill such a requirement (4Song L. Hobaugh M.R. Shustak C. Cheley S. Bayley H. Gouaux J.E. Science. 1996; 274: 1859-1866Crossref PubMed Scopus (1949) Google Scholar, 5van den Berg B. Curr. Opin. Struct. Biol. 2005; 15: 401-407Crossref PubMed Scopus (81) Google Scholar). A β barrel folds into a roughly cylindrical pore with the hydrophilic side chains oriented inside the pore lumen and the hydrophobic residues exposed to the lipid bilayer. Because the network of backbone hydrogen bonds between the neighboring β strands imparts an extraordinary stiffness to the core of the protein, β barrels are open to remodeling in various ways, including direct genetic engineering and covalent modifications (1Bayley H. Cremer P.S. Nature. 2001; 413: 226-230Crossref PubMed Scopus (965) Google Scholar, 6Bayley H. Braha O. Cheley S. Gu L.Q. Niemeyer C.M. Mirkin C.A. NanoBiotechnology. Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, Germany2004: 93-112Google Scholar, 7Movileanu L. Soft Matter. 2008; 4: 925-931Crossref PubMed Scopus (72) Google Scholar). Although redesigned β-barrel proteins are essential for stochastic biosensors, their broad application to this realm has been limited to the trimeric OmpF porins (8Nestorovich E.M. Danelon C. Winterhalter M. Bezrukov S.M. Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 9789-9794Crossref PubMed Scopus (275) Google Scholar, 9Berkane E. Orlik F. Charbit A. Danelon C. Fournier D. Benz R. Winterhalter M. J. Nanobiotechnology. 2005; 3: 3Crossref PubMed Scopus (23) Google Scholar, 10Chimerel C. Movileanu L. Pezeshki S. Winterhalter M. Kleinekathöfer U. Eur. Biophys. J. 2008; 38: 121-125Crossref PubMed Scopus (58) Google Scholar) and the heptameric α-hemolysin (αHL) 3The abbreviations used are: αHLα-hemolysinSsiemensOGn-octyl β-d-glucopyranosideDDMn-dodecyl β-d-maltosideoPOEoctyl-polyoxoethylene. pore-forming toxin (Table 1) (1Bayley H. Cremer P.S. Nature. 2001; 413: 226-230Crossref PubMed Scopus (965) Google Scholar, 3Howorka S. Siwy Z. Chem. Soc. Rev. 2009; 38: 2360-2384Crossref PubMed Scopus (941) Google Scholar, 11Howorka S. Movileanu L. Lu X.F. Magnon M. Cheley S. Braha O. Bayley H. J. Am. Chem. Soc. 2000; 122: 2411-2416Crossref Scopus (61) Google Scholar, 12Bikwemu R. Wolfe A.J. Xing X. Movileanu L. J. Phys. Condens. Matter. 2010; 22: 454117Crossref PubMed Scopus (45) Google Scholar). The multimeric character of these proteins makes them less than ideal for remodeling work (Table 1 and supplemental Fig. S1). For example, the stoichiometry and symmetry of the homomeric β-barrel pores generate many permutations and combinations of the modified (or engineered) and unmodified (wild type) monomers. This is the major reason for the technical difficulties of separating the engineered single subunit-modified protein pores with well defined biophysical and biochemical features from other products of the oligomerization reaction (13Movileanu L. Howorka S. Braha O. Bayley H. Nat. Biotechnol. 2000; 18: 1091-1095Crossref PubMed Scopus (292) Google Scholar, 14Jung Y. Bayley H. Movileanu L. J. Am. Chem. Soc. 2006; 128: 15332-15340Crossref PubMed Scopus (103) Google Scholar).TABLE 1Comparison of structural features of various β-barrel membrane proteinsProteinProtein Data Bank codeLumen occlusionsNo. of β strandsDiameterFunctional unitRefs.nmαHL7AHLNone141.5Heptameric4Song L. Hobaugh M.R. Shustak C. Cheley S. Bayley H. Gouaux J.E. Science. 1996; 274: 1859-1866Crossref PubMed Scopus (1949) Google ScholarOprD2ODJL3, L4, L7180.5Monomer67Biswas S. Mohammad M.M. Patel D.R. Movileanu L. van den Berg B. Nat. Struct. Mol. Biol. 2007; 14: 1108-1109Crossref PubMed Scopus (78) Google ScholarOpdK2QTKL3, L4, L7180.8Monomer68Biswas S. Mohammad M.M. Movileanu L. van den Berg B. Structure. 2008; 16: 1027-1035Abstract Full Text Full Text PDF PubMed Scopus (40) Google ScholarLamB1MALExtracellular loopsaInwardly folded loops (L1, L3, and L6) contribute to the constriction of ∼1/2 through the channel.180.6Trimer69Schirmer T. Keller T.A. Wang Y.F. Rosenbusch J.P. Science. 1995; 267: 512-514Crossref PubMed Scopus (531) Google ScholarOmpA1BXWNone81.0Monomer70Arora A. Rinehart D. Szabo G. Tamm L.K. J. Biol. Chem. 2000; 275: 1594-1600Abstract Full Text Full Text PDF PubMed Scopus (134) Google ScholarOmpF2OMFExtracellular loopsbLoops 1 and 4–8 partially close entrance to the lumen.,cLoop 3 folds inward and constricts the lumen.161.2Trimer71Cowan S.W. Garavito R.M. Jansonius J.N. Jenkins J.A. Karlsson R. König N. Pai E.F. Pauptit R.A. Rizkallah P.J. Rosenbusch J.P. Structure. 1995; 3: 1041-1050Abstract Full Text Full Text PDF PubMed Scopus (161) Google ScholarOmpG2F1CGating loopdLoop 6 is involved in the gating activity of the pore thereby reducing access to the lumen.142.0Monomer15Subbarao G.V. van den Berg B. J. Mol. Biol. 2006; 360: 750-759Crossref PubMed Scopus (91) Google ScholarFecA1KMOPlug (87–223) 136224.5 × 3.5eElliptical cross-sectional sides were determined using Cα positions.Monomer72Ferguson A.D. Chakraborty R. Smith B.S. Esser L. van der Helm D. Deisenhofer J. Science. 2002; 295: 1715-1719Crossref PubMed Scopus (301) Google ScholarFepA1FEBN-terminal plug (1–153) 153224.0 × 3.0eElliptical cross-sectional sides were determined using Cα positions.Monomer73Buchanan S.K. Smith B.S. Venkatramani L. Xia D. Esser L. Palnitkar M. Chakraborty R. van der Helm D. Deisenhofer J. Nat. Struct. Biol. 1999; 6: 56-63Crossref PubMed Scopus (488) Google ScholarBtuB1NQHN-terminal plug (6–132) 127224.2 × 3.7eElliptical cross-sectional sides were determined using Cα positions.Monomer74Chimento D.P. Mohanty A.K. Kadner R.J. Wiener M.C. Nat. Struct. Biol. 2003; 10: 394-401Crossref PubMed Scopus (234) Google Scholar, 75Cherezov V. Yamashita E. Liu W. Zhalnina M. Cramer W.A. Caffrey M. J. Mol. Biol. 2006; 364: 716-734Crossref PubMed Scopus (88) Google ScholarPapC3FIPVarious structuresfPlug (259–335) 77 residues.,gβ-Hairpin (447–465) 19 residues.,hα-Helix (230–240) 11 residues.244.6 × 2.8eElliptical cross-sectional sides were determined using Cα positions.Dimer76Huang Y. Smith B.S. Chen L.X. Baxter R.H. Deisenhofer J. Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 7403-7407Crossref PubMed Scopus (53) Google ScholarFhuA1BY5N-terminal plug (1–160) 160223.9 × 4.6eElliptical cross-sectional sides were determined using Cα positions.Monomer21Ferguson A.D. Hofmann E. Coulton J.W. Diederichs K. Welte W. Science. 1998; 282: 2215-2220Crossref PubMed Scopus (664) Google Scholara Inwardly folded loops (L1, L3, and L6) contribute to the constriction of ∼1/2 through the channel.b Loops 1 and 4–8 partially close entrance to the lumen.c Loop 3 folds inward and constricts the lumen.d Loop 6 is involved in the gating activity of the pore thereby reducing access to the lumen.e Elliptical cross-sectional sides were determined using Cα positions.f Plug (259–335) 77 residues.g β-Hairpin (447–465) 19 residues.h α-Helix (230–240) 11 residues. Open table in a new tab α-hemolysin siemens n-octyl β-d-glucopyranoside n-dodecyl β-d-maltoside octyl-polyoxoethylene. Recently, the outer membrane protein G (OmpG), a monomeric β-barrel pore, whose single-channel conductance and inner diameter are comparable with the corresponding values of the αHL pore (∼1 nS and ∼15 Å, respectively) (15Subbarao G.V. van den Berg B. J. Mol. Biol. 2006; 360: 750-759Crossref PubMed Scopus (91) Google Scholar), was engineered to produce a quiet unitary conductance (16Chen M. Khalid S. Sansom M.S. Bayley H. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 6272-6277Crossref PubMed Scopus (135) Google Scholar). Thus, the engineered OmpG protein might function as a nanopore-based biosensor for stochastic detection via noncovalent adaptors. However, the x-ray crystal structures of both αHL (4Song L. Hobaugh M.R. Shustak C. Cheley S. Bayley H. Gouaux J.E. Science. 1996; 274: 1859-1866Crossref PubMed Scopus (1949) Google Scholar) and OmpG (15Subbarao G.V. van den Berg B. J. Mol. Biol. 2006; 360: 750-759Crossref PubMed Scopus (91) Google Scholar) proteins reveal a somewhat small diameter of the constriction region of the pore (∼15 Å), allowing the translocation of small molecules up to ∼700 Da in molecular mass. Furthermore, the αHL, OmpF, and OmpG protein nanopores cannot permit the passage of bulky biomolecules, such as folded proteins (17Mohammad M.M. Prakash S. Matouschek A. Movileanu L. J. Am. Chem. Soc. 2008; 130: 4081-4088Crossref PubMed Scopus (95) Google Scholar, 18Mohammad M.M. Movileanu L. Eur. Biophys. J. 2008; 37: 913-925Crossref PubMed Scopus (64) Google Scholar) or even double-stranded DNA (dsDNA) (19Kasianowicz J.J. Brandin E. Branton D. Deamer D.W. Proc. Natl. Acad. Sci. U.S.A. 1996; 93: 13770-13773Crossref PubMed Scopus (2690) Google Scholar). To overcome these fundamental limitations, a larger monomeric β-barrel protein pore is required for single-molecule stochastic sensing of biomolecules, such as dsDNA, functional proteins, and their ensembles. We decided to explore the ferric hydroxamate uptake component A (FhuA), a monomeric β-barrel protein from the outer membrane of Escherichia coli. The high resolution x-ray crystal structure of FhuA is available, revealing a large membrane-spanning β-barrel domain, composed of 22 β strands (residues 161–714) (Fig. 1), which is filled by a globular N-terminal domain (residues 1–160) called the cork (20Locher K.P. Rees B. Koebnik R. Mitschler A. Moulinier L. Rosenbusch J.P. Moras D. Cell. 1998; 95: 771-778Abstract Full Text Full Text PDF PubMed Scopus (455) Google Scholar, 21Ferguson A.D. Hofmann E. Coulton J.W. Diederichs K. Welte W. Science. 1998; 282: 2215-2220Crossref PubMed Scopus (664) Google Scholar). The barrel has an elliptically shaped cross-sectional area, and the sequential β strands run anti-parallel to one another, conferring an exceptional robustness (22Bonhivers M. Desmadril M. Moeck G.S. Boulanger P. Colomer-Pallas A. Letellier L. Biochemistry. 2001; 40: 2606-2613Crossref PubMed Scopus (49) Google Scholar, 23Ramakrishnan M. Pocanschi C.L. Kleinschmidt J.H. Marsh D. Biochemistry. 2004; 43: 11630-11636Crossref PubMed Scopus (25) Google Scholar, 24Ramakrishnan M. Qu J. Pocanschi C.L. Kleinschmidt J.H. Marsh D. Biochemistry. 2005; 44: 3515-3523Crossref PubMed Scopus (41) Google Scholar). Adjacent β strands are connected by short turns on the periplasmic side and long loops on the extracellular side (Fig. 1). The x-ray crystal structure of the FhuA protein indicates that, unlike porins, the extracellular loops do not fold back into the interior of the pore but rather project away from the membrane surface (20Locher K.P. Rees B. Koebnik R. Mitschler A. Moulinier L. Rosenbusch J.P. Moras D. Cell. 1998; 95: 771-778Abstract Full Text Full Text PDF PubMed Scopus (455) Google Scholar, 21Ferguson A.D. Hofmann E. Coulton J.W. Diederichs K. Welte W. Science. 1998; 282: 2215-2220Crossref PubMed Scopus (664) Google Scholar). It has also revealed its monomeric character and elucidated the relatively clear architecture of the channel at the atomic level (Fig. 1) (20Locher K.P. Rees B. Koebnik R. Mitschler A. Moulinier L. Rosenbusch J.P. Moras D. Cell. 1998; 95: 771-778Abstract Full Text Full Text PDF PubMed Scopus (455) Google Scholar, 21Ferguson A.D. Hofmann E. Coulton J.W. Diederichs K. Welte W. Science. 1998; 282: 2215-2220Crossref PubMed Scopus (664) Google Scholar). Therefore, this information paves the way for the use of this outer membrane protein in redesign studies and in the possible development of stochastic biosensing elements. The FhuA protein exhibits a highly diverse functionality. Its primary role is to provide a binding site on the outer membrane surface for siderophores, such as ferrichrome (20Locher K.P. Rees B. Koebnik R. Mitschler A. Moulinier L. Rosenbusch J.P. Moras D. Cell. 1998; 95: 771-778Abstract Full Text Full Text PDF PubMed Scopus (455) Google Scholar, 21Ferguson A.D. Hofmann E. Coulton J.W. Diederichs K. Welte W. Science. 1998; 282: 2215-2220Crossref PubMed Scopus (664) Google Scholar, 25Pawelek P.D. Croteau N. Ng-Thow-Hing C. Khursigara C.M. Moiseeva N. Allaire M. Coulton J.W. Science. 2006; 312: 1399-1402Crossref PubMed Scopus (197) Google Scholar). In addition, FhuA also serves as a transporter of the antibiotics albomycin and rifamycin (26Ferguson A.D. Ködding J. Walker G. Bös C. Coulton J.W. Diederichs K. Braun V. Welte W. Structure. 2001; 9: 707-716Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 27Braun V. J. Bacteriol. 2009; 191: 3431-3436Crossref PubMed Scopus (44) Google Scholar), as a receptor for the antimicrobial peptide microcin J25 (MccJ25) (28Destoumieux-Garzón D. Duquesne S. Peduzzi J. Goulard C. Desmadril M. Letellier L. Rebuffat S. Boulanger P. Biochem. J. 2005; 389: 869-876Crossref PubMed Scopus (95) Google Scholar), a number of bateriophages, including T1, T5, and φ80 (29Bonhivers M. Ghazi A. Boulanger P. Letellier L. EMBO J. 1996; 15: 1850-1856Crossref PubMed Scopus (85) Google Scholar, 30Letellier L. Plançon L. Bonhivers M. Boulanger P. Res. Microbiol. 1999; 150: 499-505Crossref PubMed Scopus (53) Google Scholar, 31Lambert O. Letellier L. Gelbart W.M. Rigaud J.L. Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 7248-7253Crossref PubMed Scopus (61) Google Scholar, 32Böhm J. Lambert O. Frangakis A.S. Letellier L. Baumeister W. Rigaud J.L. Curr. Biol. 2001; 11: 1168-1175Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 33Mdzinarashvili T. Khvedelidze M. Ivanova A. Mrevlishvili G. Kutateladze M. Balarjishvili N. Celia H. Pattus F. Eur. Biophys. J. 2006; 35: 231-238Crossref PubMed Scopus (3) Google Scholar), and the protein toxin colicin M (34Cao Z. Klebba P.E. Biochimie. 2002; 84: 399-412Crossref PubMed Scopus (75) Google Scholar). Furthermore, the dynamics of the wild-type FhuA (WT-FhuA) protein at an atomistic level has been revealed by molecular dynamics simulations (35Faraldo-Gómez J.D. Smith G.R. Sansom M.S. Biophys. J. 2003; 85: 1406-1420Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). The FhuA channel exhibits a remarkable robustness, versatility, tractability, and thermal stability, as was well documented by prior spectroscopic and calorimetric studies (22Bonhivers M. Desmadril M. Moeck G.S. Boulanger P. Colomer-Pallas A. Letellier L. Biochemistry. 2001; 40: 2606-2613Crossref PubMed Scopus (49) Google Scholar, 23Ramakrishnan M. Pocanschi C.L. Kleinschmidt J.H. Marsh D. Biochemistry. 2004; 43: 11630-11636Crossref PubMed Scopus (25) Google Scholar, 24Ramakrishnan M. Qu J. Pocanschi C.L. Kleinschmidt J.H. Marsh D. Biochemistry. 2005; 44: 3515-3523Crossref PubMed Scopus (41) Google Scholar). In this study, we designed a series of single domain or multiple loop deletions to investigate which parts of the FhuA protein contribute to the occlusion of the lumen. First, we constructed a deletion mutant removing the cork domain, which encompassed the first 160 amino acids (FhuAΔ1–160) (Table 2). Second, we deleted 52% of strand β8 along with nine amino acids of loop L4 (FhuAΔ335–355). Third, we also deleted 52% of strand β8 along with most of loop L4 (FhuAΔ322–355), leaving the first seven amino acids. This construct will not have loop L4 deleted per se, but it might put a structural constraint on loop L4 to compensate for the loss of the majority of the β strand in the barrel (supplemental Fig. S2). Loop L4 is targeted for modifications, because it has been shown to reduce the extracellular entrance to the lumen of FhuA protein by ∼50% (20Locher K.P. Rees B. Koebnik R. Mitschler A. Moulinier L. Rosenbusch J.P. Moras D. Cell. 1998; 95: 771-778Abstract Full Text Full Text PDF PubMed Scopus (455) Google Scholar), and perhaps it would prevent more modulation or even the release of the cork upon the application of a transmembrane potential.TABLE 2The physical properties of the extracellular loops of the FhuA proteinLoopOverall chargeaThe total charge of the loop was calculated at pH 7.4.Charge ratiobThe total number of positive charges of the loop versus its negative charges and was calculated at pH 7.4.ResiduesLoop lengthcThe length of the loop under the stretched out conformation was based upon the total number of residues.CommentsdComments concern the cork-free FhuA protein.ÅL1−10/−1Thr170–Ser1727.0Very short loopL2+1+1/0Ala203–Ser20817.5Short loopL30+4/−4Tyr243–Asn273105Large flexible, random coil loop that folds back into the pore lumenL4+1+3/−2Cys318–His33973.5Large loop that contains three helices, and a β strand. The loop also contains a stabilizing disulfide bridge Cys318–Cys329. L4 along with part of the β strands block the access to the pore lumenL5−4+3/−7Asp394–Asn41987.5Large loop that contains a β strand, which partially occludes the pore lumenL6+1+1/0Arg463–Gly46610.5Very short loopL70+1/−1Pro502–Pro51545.5Flexible loop that does not appear to enter or block the pore lumenL8−20/−2Asp552–Phe55924.5Short loopL9+1+2/−1Asp598–Lys61145.5Medium sized flexible loop. The movement of L9 does appear to be restricted due to its positioning between two uneven β strandsL100+1/−1Gly640–Ser65449.0Medium sized flexible loop that has potential to block the pore lumenL11−2+1/−3Asn682–Arg70477.0Large loop that contains an anti-parallel β sheet, which protrudes into the pore lumena The total charge of the loop was calculated at pH 7.4.b The total number of positive charges of the loop versus its negative charges and was calculated at pH 7.4.c The length of the loop under the stretched out conformation was based upon the total number of residues.d Comments concern the cork-free FhuA protein. Open table in a new tab Previously engineered FhuA proteins were stable and functional in reconstituted systems, as judged by their channel-forming ability in planar lipid membranes (36Killmann H. Benz R. Braun V. EMBO J. 1993; 12: 3007-3016Crossref PubMed Scopus (116) Google Scholar, 37Braun V. Killmann H. Benz R. FEBS Lett. 1994; 346: 59-64Crossref PubMed Scopus (25) Google Scholar, 38Killmann H. Benz R. Braun V. J. Bacteriol. 1996; 178: 6913-6920Crossref PubMed Google Scholar, 39Braun M. Killmann H. Maier E. Benz R. Braun V. Eur. J. Biochem. 2002; 269: 4948-4959Crossref PubMed Scopus (42) Google Scholar). In the past, these deletion mutants were studied by macroscopic electrical recordings, in which detailed, time-resolved single-channel information about each deletion mutant is lacking. For example, the channel sub-states can be difficult to decipher (36Killmann H. Benz R. Braun V. EMBO J. 1993; 12: 3007-3016Crossref PubMed Scopus (116) Google Scholar, 39Braun M. Killmann H. Maier E. Benz R. Braun V. Eur. J. Biochem. 2002; 269: 4948-4959Crossref PubMed Scopus (42) Google Scholar). Thus, these macroscopic current studies hindered important conclusions about which parts of FhuA occlude the lumen. Therefore, we used single-channel electrical recordings to investigate single-deletion FhuA mutants along with the WT-FhuA protein to derive detailed information about their spontaneous, stochastic gating. In addition to single-deletion FhuA mutants, we examined double and multiple deletion mutants to obtain a comprehensive picture of the cumulative effect of both the cork domain and several large extracellular loops on the biophysical features of the FhuA protein. Based upon examination of the crystal structure of the FhuA protein (20Locher K.P. Rees B. Koebnik R. Mitschler A. Moulinier L. Rosenbusch J.P. Moras D. Cell. 1998; 95: 771-778Abstract Full Text Full Text PDF PubMed Scopus (455) Google Scholar, 21Ferguson A.D. Hofmann E. Coulton J.W. Diederichs K. Welte W. Science. 1998; 282: 2215-2220Crossref PubMed Scopus (664) Google Scholar), our major hypothesis was that L4 is not the only loop occluding the pore, but other extracellular loops may modulate the unitary conductance of the cork-free FhuA protein. We found that the removal of the cork domain and loop L4 produces an increase in the single-channel conductance up to ∼3.0 nS over WT-FhuA. In accord with our expectations, the deletion of additional extracellular loops (L3, L4, L5, and L11) resulted in a substantially enhanced single-channel conductance of ∼4.8 nS. To our knowledge, this is the highest single-channel conductance ever measured with an engineered FhuA protein (29Bonhivers M. Ghazi A. Boulanger P. Letellier L. EMBO J. 1996; 15: 1850-1856Crossref PubMed Scopus (85) Google Scholar, 36Killmann H. Benz R. Braun V. EMBO J. 1993; 12: 3007-3016Crossref PubMed Scopus (116) Google Scholar, 37Braun V. Killmann H. Benz R. FEBS Lett. 1994; 346: 59-64Crossref PubMed Scopus (25) Google Scholar, 38Killmann H. Benz R. Braun V. J. Bacteriol. 1996; 178: 6913-6920Crossref PubMed Google Scholar, 39Braun M. Killmann H. Maier E. Benz R. Braun V. Eur. J. Biochem. 2002; 269: 4948-4959Crossref PubMed Scopus (42) Google Scholar). The cork-free, multiple loop-deletion FhuA (FhuAΔC/Δ4L) proteins were either extracted from outer membranes or refolded from inclusion bodies (Fig. 1). Remarkably, high resolution single-channel electrical recordings accomplished with planar lipid bilayers showed that, although their unitary conductance is closely similar, membrane-extracted and refolded FhuAΔC/Δ4L proteins exhibit slightly different single-channel signatures. This finding pinpoints the power of single-channel electrical measurements in detecting subtle functional distinctions of membrane protein channels. pPR-IAB1 plasmids that contained wt fhua and fhuaΔ1–160, with an internal 6×His+ cloned into the coding region for the surfaced-exposed loop L5, were gifted by Professor Ulrich Schwaneberg (Jacobs University Bremen, Bremen, Germany). To construct fhuaΔ322–355, inverse PCR was performed on the wt fhua-containing plasmid with the following two phosphorylated primers: p-322, 5′-GTG ATC GAA GCT GTA GCC GAC-3′, and p-355, 5′-AAT GCT TAC AGC AAA CAG TGT-3′. The resulting PCR products were gel-purified using the MinElute® gel purification kit (Qiagen, Germantown, MD) and then self-ligated with T4 DNA ligase. To construct fhuaΔ335–355, the same strategy was applied except that p-322 was exchanged with p-335, 5′-GCG CAG GTT CTG ACG CAC AGT-3′. To construct fhuaΔ1–160/Δ322–355 and fhuaΔ1–160/Δ335–355, we applied the above overall strategy except that we performed inverse PCR on the fhuaΔ1–160-containing plasmid. All constructs were verified by DNA sequencing. The fhua gene, which lacked the regions coding for the cork domain and loops 3–5 and 11, named fhuaΔC/Δ4L, was constructed by de novo synthesis (GENEART, Regensburg, Germany) in the pMK-RQ plasmid flanked by EcoRI and XhoI restriction sites. In this construct, the deleted loops were replaced with the polypeptide NSEG(S). A serine residue was added, if it did not exist in the original loop (40Endriss F. Braun V. J. Bacteriol. 2004; 186: 4818-4823Crossref PubMed Scopus (45) Google Scholar). The pMK-RQ plasmid was digested with EcoRI and XhoI enzymes, and the released fhuaΔC/Δ4L gene was gel-purified, as mentioned above, and cloned into the pPR-IBA1 expression plasmid. This latter plasmid was also digested with EcoRI and XhoI enzymes. A C-terminal 6×His+ tag, which was preceded by a thrombin protease cleavage site, was added to fhuaΔC/Δ4L by inverse PCR utilizing the following two primers: 5′-ACT ACC GCG TGG CAG CAG AAA ACG AAA GGT TGC GGT GGC AAC-3′ and 5-CAT CAT CAC CAT CAC CAC TAA AGC GCT GGG AGC CCC CCC AGT-3′. The thrombin cleavage site and 6×His+ tag coding sequences are boldface and underlined, respectively. The final plasmid was checked by DNA sequencing. pPR-IBA1 containing the fhua gene and its derived constructs were transformed into E. coli BL21 (DE3) omp9 (F− hsdSB (rB− mB−) gal ompT dcm (DE3) ΔlamB ompF::Tn5 ΔompA ΔompC ompN::Ω (kindly provided by Dr. Helge Weingart, Jacobs University Bremen). The transformed cells were then grown in 2× TY media at 37 °C, until an A600 ∼0.7–0.8. Protein expression was induced with isopropyl β-d-1-thiogalactopyranoside, at a final concentration of 1 mm, and allowed to continue until the cell growth plateaued, as measured by A600 ∼1.4. The wild-type FhuA (WT-FhuA) protein was purified as described previously (41Nallani M. Benito S. Onaca O. Graff A. Lindemann M. Winterhalter M. Meier W. Schwaneberg U. J. Biotechnol. 2006; 123: 50-59Crossref PubMed Scopus (97) Google Scholar) with the following modifications. The outer membranes were pre-extracted in 20 mm Tris, 1 mm EDTA, 0.1% octyl-polyoxoethylene (oPOE), pH 8.0. The membrane-extracted proteins were obtained by incubating the outer membranes for 1 h at 37 °C, while shaking at 200 rpm, in 20 mm Tris, 1 mm EDTA, 3% oPOE, pH 8.0. The insoluble materials were sedimented by centrifugation at 50,000 × g for 45 min at 4 °C; the supernatant, enriched in extracted outer membrane proteins, was used for subsequent purification steps. Prior to starting purification, the detergent concentration of the solubilized WT-FhuA was reduced from 3 to 1% to lessen the effects of detergent screening during chromatographic separation. Lower concentrations of oPOE were also tested; however, the concentrations were determined to be below the critical micelle concentration and thus did not allow for complete solubilization of the WT-FhuA protein. Following the decrease of the detergent concentration, the samples were loaded onto an UNO-Q strong anion exchange column (Bio-Rad) equilibrated with 25 mm Tris, 20 mm EDTA, 1% oPOE, pH 7.8, and eluted with 250–300 mm NaCl. The FhuA-containing fractions were then pooled and concentrated (Amicon 30K MWCO). In preparation for metal affinity chromatography, the buffer was exchanged, using a Bio-Select 250–5 SEC column (Bio-Rad), to 300 mm KCl, 50 mm KH2PO4, 5 mm imidazole, 1% oPOE, pH 8.0. FhuA-containing fractions were pooled and loaded onto an immobilized metal affinity column (Bio-Rad), equilibrated with 300 mm KCl, 50 mm KH2PO4, 5 mm imidazole, 1% oPOE, pH 8.0. The column was washed with 10 mm imidazole, and the bound proteins were eluted with 250 mm imidazole, analyzed by SDS-PAGE, and used for single-channel electrical recordings (supplemental Fig. S3). Briefly, cells exp" @default.
• W1968875837 created "2016-06-24" @default.
• W1968875837 creator A5018655588 @default.
• W1968875837 creator A5023774913 @default.
• W1968875837 creator A5043821241 @default.
• W1968875837 date "2011-03-01" @default.
• W1968875837 modified "2023-09-27" @default.
• W1968875837 title "Redesign of a Plugged β-Barrel Membrane Protein" @default.
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• W1968875837 doi "https://doi.org/10.1074/jbc.m110.197723" @default.
• W1968875837 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/3048687" @default.
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