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Surface-accessible Residues in the Monomeric and Assembled Forms of a Bacterial Surface Layer Protein*

Open AccessPublished:December 01, 2000DOI:https://doi.org/10.1074/jbc.M003838200
      The S-layer protein SbsB of the thermophilic, Gram-positive organism Bacillus stearothermophilus PV72/p2 forms a crystalline, porous array constituting the outermost component of the cell envelope. SbsB has a molecular mass of 98 kDa, and the corresponding S-layer exhibits an oblique lattice symmetry. To investigate the molecular structure and assembly of SbsB, we replaced 75 residues (mainly serine, threonine, and alanine), located throughout the primary sequence, with cysteine, which is not found in the wild-type protein. As determined by electron microscopy, 72 out of 75 mutants formed regularly-structured self-assembly products identical to wild-type, thereby proving that the replacement of most of the selected amino acids by cysteine does not dramatically alter the structure of the protein. The three defective mutants, which showed a greatly reduced ability to self-assemble, were, however, successfully incorporated into S-layers of wild-type protein. Monomeric SbsB mutants and SbsB mutants assembled into S-layers were subjected to a surface accessibility screen by targeted chemical modification with a 5-kDa hydrophilic cysteine-reactive polyethylene glycol conjugate. In the monomeric form of SbsB, 34 of the examined residues were not surface accessible, while 23 were classified as very accessible, and 18 were of intermediate surface accessibility. By contrast, in the assembled S-layers, 57 of the mutated residues were not accessible, six were very accessible, and 12 of intermediate accessibility. Together with other structural information, the results suggest a model for SbsB in which functional domains are segregated along the length of the polypeptide chain.
      S-layer(s)
      bacterial cell surface layer(s)
      SLH
      S-layer homology domain
      GlcNac
      N-acetylglucosamine
      ManNAc
      N-acetylmannosamin
      NTA
      nitrilotriacetic acid
      PEG
      polyethylene glycol
      DTT
      dithiothreitol
      IVTT
      in vitro transcription/translation
      PEG-OPSS
      methoxypoly(ethylene glycol)orthopyridyl disulfide
      NEM
      N-ethylmaleimide
      PCR
      polymerase chain reaction
      PAGE
      polyacrylamide gel electrophoresis
      Bacterial cell-surface layers (S-layers)1 constitute the outermost cell envelope component of many eubacteria as well as archaea (
      • Sleytr U.B.
      • Beveridge T.J.
      ,
      • Beveridge T.J.
      ,
      • Sleytr U.B.
      • Messner P.
      • Pum D.
      • Sára M.
      ). S-layers are composed of identical protein or glycoprotein subunits, ranging in mass from 40 to 200 kDa, which have the ability to self-assemble into crystalline planar arrays. S-layer lattices can possess a variety of plane group symmetries (oblique (p1, p2), square (p4), or hexagonal (p3, p6)) and all contain pores of uniform size and morphology.
      The ultrastructure of S-layers has been well characterized by electron microscopy and some aspects of the molecular structures of certain S-layer proteins have been investigated. Most is known about the interaction of S-layer proteins with the underlying cell envelope (Refs.
      • Mesnage S.
      • Tosi-Couture E.
      • Fouet A.
      ,
      • Chami M.
      • Bayan N.
      • Peyret J.L.
      • Gulik Krzywicki T.
      • Leblon G.
      • Shechter E.
      ,
      • Bingle W.H.
      • Nomellini J.F.
      • Smit J.
      ,
      • Olabarria G.
      • Carrascosa J.L.
      • de Pedro M.A.
      • Berenguer J.
      ,
      • Dworkin J.
      • Tummuru M.K.
      • Blaser M.J.
      ,
      • Thomas S.
      • Austin J.W.
      • McCubbin W.D.
      • Kay C.M.
      • Trust T.J.
      , reviewed in Refs.
      • Sára M.
      • Sleytr U.B.
      ,
      • Engelhardt H.
      • Peters J.
      ,
      • Boot H.J.
      • Pouwels P.H.
      , and references therein). Other studies identified structurally and morphologically defined domains (
      • Thomas S.
      • Austin J.W.
      • McCubbin W.D.
      • Kay C.M.
      • Trust T.J.
      ,
      • Chu S.
      • Cavaignac S.
      • Feutrier J.
      • Phipps B.M.
      • Kostrzynska M.
      • Kay W.W.
      • Trust T.J.
      ,
      • Lupas A.
      • Engelhardt H.
      • Peters J.
      • Santarius U.
      • Volker S.
      • Baumeister W.
      ). In two cases, the surfaces of S-layers and the constituent proteins were mapped by probing the accessibility to antibodies recognizing either S-layer-specific epitopes (
      • Doig P.
      • McCubbin W.D.
      • Kay C.M.
      • Trust T.J.
      ) or a foreign epitope, which was inserted into the S-layer protein at semirandom positions (
      • Bingle W.H.
      • Nomellini J.F.
      • Smit J.
      ). Despite the relatively large number of studies, little is known about the spatial location of individual residues in assembled S-layers. Knowledge about which residues are located (i) at the external surface of the S-layer lattice, (ii) at the interface between the subunits, and (iii) within the pores (Fig.1) is crucial for a better understanding of the assembly and molecular structure of S-layers.
      Figure thumbnail gr1
      Figure 1Schematic drawing of a top view (A) and a cross-sectional (B) view of an oblique S-layer bound on the cell wall. The cross-sectional view depicts the lattice of the top view along the drawn line. The S-layer protein subunit, the outer and inner surface of the S-layer lattice, the interface between the subunits, and the pores are indicated.
      In this report, cysteine scanning mutagenesis combined with targeted chemical modification (
      • Altenbach C.
      • Marti T.
      • Khorana H.G.
      • Hubbell W.L.
      ,
      • Akabas M.H.
      • Stauffer D.A.
      • Xu M.
      • Karlin A.
      ,
      • Walker B.
      • Bayley H.
      ,
      • Frillingos S.
      • Sahin-Toth M.
      • Wu J.
      • Kaback H.R.
      ) is used to identify surface accessible residues (
      • Krishnasastry M.
      • Walker B.
      • Braha O.
      • Bayley H.
      ) in SbsB. SbsB is the S-layer protein of Bacillus stearothermophilus PV72/p2, a thermophilic Gram-positive bacterium (
      • Sára M.
      • Sleytr U.B.
      ,
      • Kuen B.
      • Koch A.
      • Asenbauer E.
      • Sára M.
      • Lubitz W.
      ). SbsB has a molecular mass of 98 kDa and assembles into a lattice of oblique symmetry. The N terminus of SbsB (residues 34 to 212 in the preprotein sequence) encodes three S-layer homology (SLH)1 domains (
      • Engelhardt H.
      • Peters J.
      ). SLH domains were originally identified by sequence comparison (
      • Lupas A.
      • Engelhardt H.
      • Peters J.
      • Santarius U.
      • Volker S.
      • Baumeister W.
      ) and have been shown to attach exoproteins of Gram-positive bacteria to the underlying cell wall (see Refs.
      • Sára M.
      • Sleytr U.B.
      and
      • Engelhardt H.
      • Peters J.
      , and references therein). In most proteins studied so far, SLH domains specifically bind to secondary cell wall polymers (
      • Sára M.
      • Sleytr U.B.
      ), and, in the case of SbsB, the polymer is composed mainly of GlcNAc and ManNAc (
      • Ries W.
      • Hotzy C.
      • Schocher I.
      • Sleytr U.B.
      • Sára M.
      ). Apart from binding the secondary cell wall polymer (
      • Sára M.
      • Sleytr U.B.
      ), the N terminus of SbsB also binds to the peptidoglycan-containing cell wall itself (
      • Sára M.
      • Egelseer E.M.
      • Dekitsch C.
      • Sleytr U.B.
      ). In addition, SbsB contains a second binding region for the secondary cell wall polymer, which was mapped to amino acid positions 271–362 (
      • Sára M.
      • Egelseer E.M.
      • Dekitsch C.
      • Sleytr U.B.
      ). Under native conditions, the N terminus (up to amino acid position 206) of SbsB can be removed by limited proteolysis, while the remainder of the SbsB polypeptide is proteolytically stable.
      Y. Wang and H. Bayley, manuscript in preparation.
      2Y. Wang and H. Bayley, manuscript in preparation.
      A shortened version of SbsB, beginning with amino acid 200, is sufficient for S-layer formation, as studies with genetically truncated versions of SbsB have demonstrated.2 Deletions of the C terminus, however, abolish self-assembly. These data suggest the existence of a biochemically defined structural domain, which might correspond to the massive morphological domain, visible in computer-processed transmission electron micrographs of S-layer lattices from B. stearothermophilus PV72/p2 (

      Ballesteros, J. A., Deupi, X., Olivella, M., Haaksma, E. E. J., and Pardo, L. (2000) Biophys. J., in press.

      ).
      The goal of the present study was to use targeted chemical modification to identify residues, which are located at the surface of the S-layer protein SbsB. By using monomeric SbsB and SbsB assembled on cell walls, we have been able to discriminate between residues which are (i) located at the surface of the monomer, (ii) positioned on the outer surface of the S-layer lattice or within the lumen of the pores, and (iii) located on the inner surface of the lattice or at the interface between assembled subunits (Fig. 1). In addition, the availability of the extensive set of surface-located cysteines will facilitate further investigations of SbsB and may allow the engineering of S-layers by targeted chemical modification to produce novel biomolecular materials.

      RESULTS

      The properties of 75 cysteine mutants of SbsB are reported here. With the exception of mutant R79C, the mutations were dispersed throughout the primary sequence between amino acid positions 234 and 913 of the 920-residue SbsB polypeptide. The numbers refer to the amino acid positions in the sequence of the preprotein (
      • Kuen B.
      • Koch A.
      • Asenbauer E.
      • Sára M.
      • Lubitz W.
      ). All mutants were derived from Wt-SbsB-His, which lacks the 31-amino acid signal peptide, but carries a 6xHis tag at the C terminus attached through a 15-amino acid linker. The sequence of Wt-SbsB-His differs from the natural sequence at three codons altered by PCR errors: Thr210 → Ser, Ser402 → Thr, Lys812 → Glu. Electron microscopy showed that neither the C-terminal 6xHis tag nor the three point mutations changed the ability of the S-layer protein to self-assemble (Fig. 3 B). In the following, Wt-SbsB-His refers to this sequence. The S-layer protein SbsB isolated fromB. stearothermophilus PV72/p2 is referred to as Wt-SbsB.
      Figure thumbnail gr3
      Figure 3Electron micrographs of negative-stained self-assembly products of recombinantly expressed mutant T315C (A) and Wt-SbsB-His (B). Bars, 150 nm.
      For the scanning mutagenesis, the residues alanine, serine, and threonine were the primary choices for the mutation to cysteine, thereby producing conservative changes that would not disrupt the native structure of SbsB. This selection was also intended to increase the likelihood of finding surface accessible residues, since polar residues, such as serine and threonine, tend to be located at the surface of proteins (
      • Miller S.
      • Janin J.
      • Lesk A.M.
      • Chothia C.
      ,
      • Tsai C.J.
      • Lin S.L.
      • Wolfson H.J.
      • Nussinov R.
      ).
      All 75 mutants were generated by site-directed mutagenesis using a PCR-based protocol featuring a low number of PCR cycles. Sequencing of single isolates of two different mutants showed that no PCR errors had occurred. To ease the identification of positive clones, anNsiI restriction site (ATGCAT), which encompassed the cysteine codon (underlined), was introduced with each mutation.
      For all mutants at least two independent isolates were generated. For the biochemical assays on the thermal stability of monomeric cysteine mutants in SDS, and on the surface accessibility of monomeric and assembled cysteine mutants by PEG-OPSS modification, two independent isolates for each mutation were used. For the assay on the ability of the cysteine mutants to self-assemble as determined by negative staining and electron microscopy, one isolate was used, unless otherwise stated.

      In Vivo Expression and Self-assembly of SbsB Cysteine Mutants

      SDS-PAGE confirmed that His-tagged SbsB mutants were expressed at high levels in vivo (Fig.2 A, lane 1) and could be quantitatively removed from the cell lysate by binding to Ni-NTA resin, as exemplified by mutant G273C (Fig. 2 A, lane 2). All mutants were expressed and purified, yielding similar protein concentrations in the eluates from Ni-NTA resin, as shown for 13 different mutants (Fig. 2 B). The average total protein yield after three elutions was approximately 8 mg of SbsB protein from 4.5 ml of bacterial culture. The purified mutants were all dialyzed under the same conditions. During dialysis a white precipitate formed. To test whether self-assembly products had been formed, the precipitates of all 75 mutants were analyzed by electron microscopy following negative staining. For example, the electron micrograph of the precipitate from mutant T315C (Fig. 3 A) shows a regular pattern with oblique lattice symmetry characteristic of Wt-SbsB-His self-assembly products (Fig. 3 B). In summary, 72 out of 75 mutants had the ability to form self-assembly products like Wt-SbsB-His (Table I). Therefore, in most cases, the mutation does not dramatically alter the structure of the protein, even though the substitution to cysteine is often accompanied by a second, conservative
      The following amino acid changes were considered conservative: alanine, serine, threonine to cysteine; alanine, leucine, phenylalanine, tyrosine, valine to isoleucine; aspartic acid to glutamic acid; alanine to methionine; phenylalanine to leucine. All other amino acid changes were non-conservative.
      amino acid replacement to allow the introduction of an NsiI restriction site (ATGCAT; cysteine codon underlined). In some cases non-conservative amino acid replacements were deliberately generated to probe the conformational tolerance of SbsB (e.g. E837C/Q838I). In summary, among the 48 cysteine mutants, which were accompanied by a second amino acid change, 29% (14/48) had one conservative and one non-conservative change, 2% (1/48) two non-conservative changes while the rest, 69% (33/48), had two conservative amino acid changes. Among the 18 mutants with amino acid changes in three positions, 6% (1/18) were conservative in all three positions, 55% (10/18) conservative in two positions, and 39% (7/18) conservative in one position. 56% (5/9) of cysteine mutations not accompanied by a second change were non-conservative. The three isolates that did not show self-assembly products with regular lattices were: T240C/V241I; D723E/G724C, and T816C/G817I. These three isolates did, however, form a white precipitate upon dialysis. To exclude the possibility of additional mutations stemming from defective mutagenic primers, we sequenced the corresponding region and found no errors. The electron microscopic examination of second and third isolates confirmed that the three mutants were assembly-compromised, and this eliminates the possibility that mutations stemming from PCR errors might have caused the assembly-compromised phenotype. It was, however, observed, that in one out of two separate dialysates of one isolate of mutant T240C/V241I traces of S-layer formed, suggesting that the ability to assemble had not been completely eliminated in this mutant.
      Figure thumbnail gr2
      Figure 2Expression and Ni-NTA affinity chromatographic purification of SbsB cysteine mutants analyzed by Coomassie-stained SDS-polyacrylamide gels. A, lane 1, urea-solubilized fraction of E. coli cells harboring plasmid pT7sbsBHis-G273C; lane 2, flow-through after incubation of the cell lysate with Ni-NTA resin. B, first eluates of 6xHis tag purified SbsB mutants (2 μl from 150 μl).Lane 1, T492C; lane 2, K486C; lane 3, S480C; lane 4, S714C; lane 5, T708C; lane 6, V698C; lane 8, Y691C; lane 9, T683C;lane 10, R674C; lane 11, S662C; lane 12, S651C; lane 13, D505C. Lane 7, S894C (2.5 μl) which had been isolated in a different purification. Thearrow indicates the protein band of SbsB.
      Table IProperties of single cysteine mutants of SbsB
      In vitro expressed
      Monomeric formIncorporated into self-assembly products
      In vivo expressed, Self-assemblyThermal stability in SDSModificationModification
      Wt-SbsB-His++++
      R79C/V80I+++++
      F233L/A234C+++++
      T240C/V241I+++++++
      T245C/L246I++++
      S252C/V253I++++
      F261L/T262C/R263I++++++++
      T270C/A271M+++++
      G273C++++++++
      T281C/A282I+++++++
      F289C/V290I++++
      T298C/V299I++++
      N303K/S304C/S305M++++
      T315C/V316I+++++
      G326C/A327I+++++
      S333C/S334I++
      S347C/V348I++++++++
      T354C/A355I++++
      T368C/L369I++++
      N377K/T378C/F379I+++++
      T382C/V383I++++
      N407K/T408C/V409I++++
      S419C++++
      T427C/V428I++
      D432E/T433C/K434I++++++++++
      T440C/K441I++
      T449C/V450I+++++++
      T459C/A460I+++
      T470C/A471I+++
      S480C/F481I++++++++
      F485L/K486C/D487I++++++
      T492C/F493I+++++
      D505C++++++++
      S512C/V513I++++
      A519C/V520I++++
      T532C++++
      E544C++++++++
      T554C/V555I+++++++
      V563C++++
      N571K/T572C++++
      N579K/S580C/A581I++++++++
      I594C/V595I++++
      N606K/A607C/S608I++++++++
      T617C+++++
      D628E/A629C/T630I+++++
      S640C/E641I++++
      N645C/A646I+
      S651C/T652I++++++++
      S662C/V663I+++++
      N673K/R674C/L675I+++
      T683C/T684I++++
      D690E/Y691C/V692I++++
      N697K/V698C/L699I++++
      T708C/L709I++
      S714C/T715I+++++
      D723E/G724C++++
      S738C/V739I++++
      T744C/V745I++++++++
      T751C/V752I+++++
      T762C/F763I++
      F768L/G769C/V700I+
      D781C++++++++
      A791C/A792I+++++
      D795E/G796C/Y797I++
      T805C/V806I+++
      T816C/G817I
      D826E/A827C/T828I++
      E837C/Q838I+++++++
      F845L/S846C/D847I+
      F854L/S855C/L856I+++++++
      T866C/V867I++++++++
      D876E/A877C++++
      S887C/F888I+++++++
      S894C++++
      N905K/N906C/L907I++
      S913C/V914I+++++++
      The properties of in vivo expressed mutants (self-assembly) and in vitro expressed mutants (thermal stability in SDS and surface accessibility) are tabulated. The assays for in vitroexpressed mutants were performed for two independent isolates, and the assay for in vivo expressed mutants on one isolate, unless otherwise stated. S-layer formation was determined by electron microscopy. The assay of the thermal stability in SDS is a tool to probe the conformation of the monomeric mutant based on the structural stability of an SDS-refractile folded motif. Modification with PEG-OPSS was performed for monomeric cysteine mutants and for SbsB cysteine mutants incorporated into self-assembly products of SbsB from B. stearothermophilus PV72/p2 bound to peptidoglycan-containing cell wall sacculi of B. stearothermophilus PV72/p2. Key: self-assembly: +, self-assembly products similar to Wt-SbsB-His; −, greatly reduced ability to form self-assembly products (confirmed with three independent isolates). Thermal stability in SDS: +++, at 25 °C, 80-kDa SbsB band and at 50 °C, 80- and 100-kDa bands at similar intensities (i.e. like Wt-SbsB-His); +, at 25 °C, 80-kDa band and at 50 °C, 100-kDa band predominates; −, 100 kDa band at 25 and 50 °C. Modification: +++, more than 50% modification after 20 s reaction; +, between 50 and 10% modification after 20 s; −, less than 10% modification after 20 s. 100% modification is the extent of modification for the denatured protein.

      In Vitro Expression of Cysteine Mutants

      Selected SbsB cysteine mutants
      In the following, mutants are referred to only by the amino acid, which was mutagenized to cysteine (e.g. T240C/V241I is T240C).
      were expressed in vitro with radiolabeled cysteine to demonstrate the successful mutational change to cysteine. For example, G273C was expressed in vitro with [35S]cysteine and analyzed by SDS-PAGE and autoradiography (Fig.4). As expected, G273C (Fig. 4,lane 3) but not Wt-SbsB-His (Fig. 4, lane 1), which does not harbor a cysteine codon, gave a positive signal. With [35S]methionine both Wt-SbsB-His (Fig. 4, lane 2) and G273C (Fig. 4, lane 4) were labeled. (Wt-SbsB-His contains 5 methionine codons.) The major protein band migrating at 100 kDa (indicated by an arrow in Fig. 4) corresponds to full-length SbsB. The less intense and faster migrating bands most likely result from premature termination of transcription or translation, since they did not bind to Ni-NTA resin (data not shown), suggesting that they did not contain the C-terminal 6xHis tag.
      Figure thumbnail gr4
      Figure 4Incorporation of radiolabeled cysteine into the in vitro - expressed cysteine mutant SbsB G273C. Supercoiled plasmid DNA pT7sbsBHis (lanes 1 and 2) and pT7sbsBHis-G273C (lanes 3 and 4) were used for in vitro expression with either [35S]cysteine (lanes 1 and3) or [35S]methionine (lanes 2 and4). The arrow indicates the protein band of full-length SbsB.

      Definition of the Different Substrates Used for PEG-OPSS Modification

      The assembly states of in vitro expressed SbsB and Wt-SbsB self-assembly products doped with in vitroexpressed SbsB were examined by size exclusion chromatography and electron microscopy, respectively.
      To determine whether in vitro-expressed SbsB was monomeric or oligomeric, in vitro-expressed [35S]methionine-labeled Wt-SbsB-His was subjected to size exclusion chromatography in 50 mm Na phosphate buffer, pH 7.0, containing 0.15 m NaCl. Analysis of the fractions by SDS-PAGE and autoradiography (not shown) revealed that Wt-SbsB-His (M r 100,000) eluted between the marker proteins aldolase (M r 158,000) and albumin (M r 66,000). This result is consistent with the notion, that in vitro-expressed Wt-SbsB-His is monomeric.
      All 75 in vitro-expressed SbsB mutants (two independent isolates each) were incorporated into S-layers by dialyzing His-tag purified [35S]methionine-labeled SbsB out of 8m urea in the presence of a 5000-fold molar excess ofin vivo expressed Wt-SbsB and cell wall sacculi of B. stearothermophilus PV72/p2. Self-assembly products of Wt-SbsB bind with their inner surface against the peptidoglycan-containing cell wall sacculi leaving the outer side of the lattice surface exposed and accessible (
      • Ries W.
      • Hotzy C.
      • Schocher I.
      • Sleytr U.B.
      • Sára M.
      ) (Fig. 1). In the present study, cell wall-bound self-assembled mutants were used to identify residues located on the outer surface of the S-layer. It was undesirable to use S-layers assembled in the absence of cell wall sacculi, because free S-layers tend to form double layers in suspension. An electron micrograph shows that Wt-SbsB assembled onto peptidoglycan-containing cell wall sacculi as sheet-like S-layers (Fig. 5). Rigorous examination of the ultrathin sections of embedded sacculi confirmed that the preparations did not contain unbound S-layers. Free S-layer monolayers and multilayers were found, however, when the ratio of Wt-SbsB protein over cell-wall sacculi was increased (not shown).
      Figure thumbnail gr5
      Figure 5Electron micrographs of ultrathin-sectioned self-assembly products of SbsB from B. stearothermophilusPV72/p2 bound to peptidoglycan-containing cell wall sacculi ofB. stearothermophilus PV72/p2. Self-assembly products (s) and peptidoglycan-containing cell wall sacculi (pg) are indicated. Most cell-wall sacculi carry the S-layer on the outer side, while in a few cases patches of S-layer also assembled onto the inner side. B. stearothermophilus cells are rod-shaped bacteria, hence the micrograph depicts circular, transversally cut (center of picture), and oval, longitudinally cut cell wall sacculi (upper left) in addition to small cell wall fragments. Bar,500 nm.
      Since 72 in vivo-expressed SbsB cysteine mutants were able to crystallize into self-assembly products like Wt-SbsB-His (see above), it was very likely that in vitro expressed SbsB cysteine mutants would crystallize with Wt-SbsB to form S-layers. To determine how much in vitro expressed SbsB was incorporated into self-assembly products, we determined the amount of radiolabeled SbsB in the supernatant and the pellet after dialysis for all mutants. Analysis by SDS-PAGE and PhosphorImager quantification demonstrated that more than 90% of the in vitro expressed SbsB became incorporated into self-assembly products with Wt-SbsB (data not shown). The fraction of incorporated in vitro expressed protein was very similar for all 75 mutants tested. This indicates that thein vitro expressed mutants T240C, G724C, and T816C were incorporated into the lattice of Wt-SbsB. By contrast, the in vivo expressed mutant polypeptides had a greatly reduced ability to form crystalline self-assembly products in the absence of Wt-SbsB. Hence, it is likely that SbsB mutants T240C, G724C, and T816C adopt the assembly-competent conformation in the presence of a large excess of wild-type protein. In the following, T240C, G724C, and T816C were included in the assays designed to probe the structure of SbsB.

      Assay of the Thermal Stability of Monomeric Cysteine Mutants in SDS

      This assay was performed to probe the conformation of the [35S]methionine-labeled monomeric form of the mutants and carried out for all 75 mutants with two independent isolates. Wt-SbsB-His (like Wt-SbsB) remains partly folded in SDS at 25 °C. Upon SDS-PAGE, the unheated protein migrates at an apparent molecular mass of 80 kDa (Fig. 6 A, lane 1) compared with 100 kDa for heated unfolded SbsB (Fig. 6 A, lane 3). After treatment at 50 °C, both the partly folded, faster migrating form as well as the unfolded form were found at similar band intensities (Fig. 6 A, lane 2). As an example, this analysis is shown for mutants T440C (Fig. 6 A, lanes 4–6), S640C (Fig. 6 A, lanes 7–9), and N645C (Fig.6 A, lanes 10–12) with three different incubation temperatures (25, 50, and 100 °C). At an incubation temperature of 25 °C, mutant S640C (Fig. 6 A, lane 7) and Wt-SbsB-His (Fig. 6 A, lane 1) showed one major protein band migrating at an apparent molecular mass of 80 kDa corresponding to an partly unfolded polypeptide chain. When the incubation temperature was changed to 50 °C, S640C (Fig. 6 A, lane 8) and Wt-SbsB-His (Fig.6 A, lane 2) exhibited two major protein bands migrating at apparent molecular masses of 80 and 100 kDa. At 100 °C, the major protein band of S640C (Fig. 6 A, lane 9) and Wt-SbsB-His (Fig. 6 A, lane 3) migrated at 100 kDa. The similarities in the temperature-dependent migration of S640C and Wt-SbsB-His are consistent with the notion that the mutations in S640C do not destabilize the structural motif in SDS. In contrast, the major protein band of N645C runs at 100 kDa at 25 °C and 50 °C (Fig.6 A, lanes 10 and 11, respectively) indicating that this mutant is more readily unfolded in SDS than Wt-SbsB-His. T440C exhibited a 80-kDa band at 25 °C and only one band migrating at 100 kDa at 50 °C (Fig. 6 A, lanes 4 and 5, respectively). This suggests that T440C is less readily unfolded in SDS than N645C, but more readily unfolded than Wt-SbsB-His. In summary, 51 out of 75 mutants showed a behavior similar to Wt-SbsB-His. Six mutants had a strongly destabilized phenotype (only a 100-kDa band independent of the incubation temperature) and 18 mutations were moderately destabilized (80-kDa band at 25 °C and a 100-kDa band at 50 °C) (Table I).
      Figure thumbnail gr6
      Figure 6Examination of the thermal stability of SbsB mutant monomers and the surface accessibility of the cysteine sulfhydryls of SbsB mutant monomers and SbsB mutants incorporated into self-assembly products of SbsB from B. stearothermophilusPV72/p2. A, assay of the thermal stability in SDS of [35S]methionine-labeled in vitro expressed, monomeric cysteine mutants of SbsB. After in vitroexpression the IVTT reaction was mixed with Laemmli buffer and incubated for 5 min at 25, 50, or 100 °C before loading onto the gel. Lanes 1–3, WT-SbsB-His; lanes 4–6, T440C;lanes 7–9, S640C; lanes 10–12, N645C. The increasing incubation temperature is indicated by atriangle, with 25 °C at the left, 50 °C in the middle, and 100 °C at the right end of thetriangle. The bands of faster migrating, partly unfolded, and slower migrating, unfolded SbsB are indicated by arrows. B and C, PEG-OPSS modification of in vitroexpressed SbsB cysteine mutants to determine the surface accessibility of the cysteine sulfhydryls in B, the SbsB monomer, and inC, SbsB incorporated into self-assembly products of Wt-SbsB from B. stearothermophilus PV72/p2 bound to peptidoglycan-containing sacculi of B. stearothermophilusPV72/p2. In vitro expressed cysteine mutants T683C, T617C, S580C, and S855C (either monomeric or incorporated into self-assembly products were mixed with the sulfhydryl-reactive reagent PEG-OPSS and incubated for 10 s (lanes 1, 4, 7, and 10) or 20 s (lanes 2, 5, 8, and 11) at 0 °C before the reaction was terminated by the addition ofN-ethylmaleimide. The extent of modification of unfolded protein was obtained by boiling a reaction mixture for 5 min in the presence of SDS before the addition of NEM (lanes 3, 6, 9, and 12). Lanes 1–3, T683C; lanes 4–6, T617C; lanes 7–9, S580C; lanes 10–12, S855C. The positions of the unmodified, not shifted and modified, upshifted SbsB bands are indicated byarrows.
      Although the assay of the thermal stability of cysteine mutant monomers indicates that some mutations affect the conformational stability in SDS, all except three mutants retained their ability to assemble into crystalline arrays under native conditions (see above). Mutant T816C was both compromised in assembly and destabilized in SDS. However, two of the three mutants compromised in assembly did not have a destabilized phenotype in SDS (T240C and G724C). Therefore, a destabilized phenotype is not required to interfere with self-assembly.

      Surface Accessibility of Monomeric Cysteine Mutants by PEG-OPSS Modification

      To assess the surface accessibility of the cysteine residues in the folded monomers in solution, all 75 in vitroexpressed [35S]methionine-labeled SbsB cysteine mutants (two independent isolates of each) were subjected to the sulfhydryl-specific PEG-OPSS modification. The modification reagent PEG-OPSS has a molecular mass of approximately 5 kDa and is therefore expected to react preferentially with sterically unhindered, surface-located cysteines, whereas the reaction with residues buried within the SbsB protein would be restricted due to steric exclusion. PEGylation of SbsB results in an increase of the molecular mass, which can be monitored by SDS-PAGE. Interestingly, modification with PEG-OPSS with a molecular mass of 5 kDa resulted in a gel shift corresponding to an increase of approximately 15 kDa in apparent molecular mass. An example of this type of analysis is displayed in Fig. 6 B for mutants T683C (Fig. 6 B, lanes 1–3), T617C (Fig. 6 B, lanes 4–6), S580C (Fig. 6 B, lanes 7–9), and S855C (Fig. 6 B, lanes 10–12). The extent of modification was determined for reaction times of 10 s (Fig.6 B, lanes 1, 4, 7, and 10) and 20 s (Fig.6 B, lanes 2, 5, 8, and 11) under nondenaturing conditions at 0 °C, and for 5 min in the presence of SDS at 100 °C (Fig. 6 B, lanes 3, 6, 9, and 12). The reactions for 10 and 20 s were performed at 0 °C to detect differences in the rate of modification; the reaction at room temperature results in almost instant modification (data not shown). The modification reaction was stopped by the addition of sulfhydryl-reactive NEM in 56 molar excess over PEG-OPSS. When mutant T683C was allowed to react for 10 s (Fig. 6 B, lane 1) and 20 s (Fig. 6 B, lane 2) with the reagent, the major protein band migrated at 100 kDa, showing that no modification had occurred. However, under denaturing conditions the major gel band was upshifted (Fig. 6 B, lane 3) due to the covalent modification with PEG. The modification pattern for mutant T683C under native and denaturing conditions suggests that the cysteine residue at position 683 is not surface accessible in the native state. In contrast, mutants S580C and S855C exhibited both an unmodified and an upshifted protein band after the 10-s reaction time under native conditions (Fig.6 B, lanes 7 and 10, respectively). After 20 s, the extent of modification was increased (Fig. 6 B, lanes 8 and 11). Under denaturing conditions, mutants S580C and S855C became completely modified and migrated as upshifted band (Fig. 6 B, lanes 9 and 12). The patterns of modification indicate that cysteine residues at positions S580C and S855C are surface accessible. Mutant T617C exhibited a reactivity pattern, which was consistent with an intermediate surface accessibility (Fig. 6 B, lanes 4–6). In summary, 34 mutants were not surface accessible, 23 were very accessible, and 18 were of intermediate surface accessibility as probed by the modification with PEG-OPSS (Table I). The upshift of PEGylated SbsB could be reversed by the addition of DTT, which cleaves the disulfide bridge between the protein and PEG (data not shown). This proved that the SbsB mutants were specifically PEGylated at cysteine.

      Surface Accessibility of SbsB Cysteine Mutants in S-layers by PEG-OPSS Modification

      To probe the surface accessibility of assembled cysteine mutants, all 75 in vitro-expressed [35S]methionine-labeled SbsB cysteine mutants (two independent isolates of each) were incorporated into Wt-SbsB S-layers bound to peptidoglycan-containing cell wall sacculi of B. stearothermophilus PV72/p2. Then, the samples were subjected to the sulfhydryl-specific PEG-OPSS modification and analyzed by SDS-PAGE. As an example, this analysis is shown for mutants T683C (Fig. 6 C, lanes 1–3), T617C (Fig. 6 C, lanes 4–6), S580C (Fig.6 C, lanes 7–9), and S855C (Fig. 6 C, lanes 10–12). The extent of modification was determined for reaction times of 10 s (Fig. 6 C, lanes 1, 4, 7, and10) and 20 s (Fig. 6 C, lanes 2, 5, 8, and 11) under nondenaturing conditions at 0 °C, and for 5 min in the presence of SDS at 100 °C (Fig. 6 C, lanes 3, 6, 9, and 12). The assembled mutant T683C remained unmodified after 10 and 20 s (Fig. 6 C, lanes 1 and2, respectively), but could be modified under denaturing conditions (Fig. 6 C, lane 3). This suggests that cysteine in position 683 is not surface accessible in the assembled form; the cysteine in monomeric T683C was not surface accessible either (Fig.6 B, lanes 1–3). Interestingly, the assembled mutant T683C could not be completely modified under denaturing conditions (Fig.6 C, lane 3), which is in contrast to the complete modification of T683C monomers (Fig. 6 B, lane 3). The incomplete modification of the assembled mutant might indicate that irreversible oxidation of the sulfhydryl group of cysteine occurred in the course of the 2-day-long dialysis of the samples, even though dialysis was performed under reducing conditions and DTT was added prior to modification. Incomplete modification under denaturing conditions was also observed for all other assembled mutants (e.g. Fig. 6 C, lanes 3, 6, 9, and 12). Therefore, the extent of modification under native conditions was normalized with respect to the extent of modification obtained under denaturing conditions. Assembled mutant S580C showed modification after 10 s (Fig. 6 C, lane 7) and reached maximal modification after 20 s (Fig. 6 C, compare lane 8 withlane 9). This indicates that the cysteine at position 580, which was very surface accessible in the monomer (Fig. 6 B, lanes 7–9), retains its high surface accessibility when assembled into S-layers. In contrast, the cysteine in mutant S855C, which was very surface accessible in the monomer (Fig. 6 B, lanes 10–12) was not modified in the assembled form (Fig. 6 C, lanes 10–12). Similarly, the cysteine residue in mutant T617C was occluded in the self-assembly product (Fig. 6 C, lanes 4–6), while it exhibited intermediate surface accessibility in the monomeric form (Fig. 6 B, lanes 4–6). In summary, 57 assembled mutants were not surface accessible, six were very surface accessible, and 12 were of intermediate surface accessibility as probed by the modification with PEG-OPSS (Table I).

      DISCUSSION

      To better understand the molecular structure and assembly of S-layer proteins, it is important to know which residues are located (i) at the external surface of the S-layer lattice, (ii) at the interface between the subunits, and (iii) within the pores (Fig. 1). In the present study, the differential accessibility of monomeric and assembled SbsB, the S-layer protein of B. stearothermophilusPV72/p2, was investigated. Seventy-five single-cysteine mutants were generated and analyzed by targeted chemical modification to probe the surface accessibility of the cysteine residues. Seventy-four mutations were evenly dispersed throughout the primary sequence between amino acid positions 234 and 913 of the 920-residue SbsB polypeptide (Fig.7 A, black bars). The N-terminal 200 amino acids were not included in the scanning mutagenesis, because this portion of SbsB can be deleted without affecting the formation of self-assembly products2 (Fig.7 B).
      Figure thumbnail gr7
      Figure 7Graphical summary of the most important features of single-cysteine substitution mutants of SbsB correlated with other structural information. A, chart displaying the results of the analysis of in vivo andin vitro expressed SbsB cysteine mutants. Key: Blue bars, reduced ability to form assembly products as assayed by electron microscopy. Small and large green bars, respectively, moderately and strongly reduced stability of in vitro expressed SbsB monomers in SDS. Small andlarge red bars, respectively, very high surface accessibility of cysteine residues in monomers and in self-assembly products bound to cell wall sacculi. Black bars, cysteine mutants analyzed in this work. Small bars, single mutation;medium bar, double mutation; large bars, triple mutation. The positions of the cysteine residues refer to their positions in the unprocessed SbsB polypeptide carrying the signal sequence. B, effect of N- and C-terminal deletions of recombinantly expressed SbsB on the formation of self-assembly products as assayed by electron microscopy.2 C, schematic drawing of SbsB, showing the location of the S-layer homology (SLH) domain, and the peptidoglycan and secondary cell wall polymer (SCWP)-binding domains. The SLH domains were found by sequence comparison (
      • Engelhardt H.
      • Peters J.
      ), and the cell wall-binding domains were derived from proteolysis protection assays, and binding assays with proteolytically and genetically truncated fragments of SbsB. (Amino acid positions are given in parentheses and refer to the preprotein polypeptide sequence.) Adapted from Refs.
      • Sára M.
      • Sleytr U.B.
      and
      • Sára M.
      • Egelseer E.M.
      • Dekitsch C.
      • Sleytr U.B.
      .
      To test whether point mutations to cysteine affect the conformation of SbsB, we first examined the formation of self-assembly products byin vivo expressed protein. Electron microscopic examination revealed that mutants T240C, G724C, and T816C had a greatly reduced ability to form regular lattices under conditions where the other mutants self-assembled. The positions of these three mutations are indicated in Fig. 7 A (blue bars).
      Next, we probed the thermal stability of an SDS-refractile structural motif in in vitro expressed monomeric protein, which causes SbsB to migrate at an apparent molecular mass of 80 kDa upon SDS-PAGE (Fig. 6 A). The unidentified structural motif is most likely a β-sheet, since other β-sheet-rich proteins often exhibit a similar behavior (
      • Nakamura K.
      • Mizushima S.
      ,
      • Klose M.
      • Schwarz H.
      • MacIntyre S.
      • Freudl R.
      • Eschbach M.L.
      • Henning U.
      ,
      • Kleinschmidt J.H.
      • Wiener M.C.
      • Tamm L.K.
      ). Apparently, the structural motif includes the C terminus of SbsB, as C-terminal truncation mutants2and prematurely terminated in vitro expressed SbsB (Fig.6 A, lanes 1–3) do not exhibit temperature-dependent gel migration. In summary, of 75 mutants analyzed, 51 showed a behavior similar to Wt-SbsB-His (Table Iand Fig. 7 A). Six mutants had a strongly destabilized phenotype (only a 100-kDa band independent of the incubation temperature) and 18 mutants were moderately destabilized (80-kDa band at 25 °C and a 100-kDa band at 50 °C). All except one (T816C) destabilized mutants formed self-assembly products, indicating that the mutants folded correctly but were unfolded in the presence of SDS. Correlation of the observed phenotype with the severity of the mutation (conservative or non-conservative mutations; single, double, or triple mutations) revealed a bias toward the destabilized phenotype when two or more amino acids were changed. Among triple mutations 17% (3 out of 18) were strongly destabilizing and 39% (7/18) moderately destabilizing, as compared with 6% (3/48) and 21% (10/48) of double mutations, and 0% and 11% (1/9) of single mutations. The positions of the different classes of mutations were, however, scattered throughout the primary sequence of SbsB (Table I and Fig. 7 A). This allowed us to consider structure-destabilizing mutations with regard to their position in the primary sequence. All six mutants with a strongly decreased stability in SDS were clustered toward the C terminus of SbsB: N645C, G769C, G796C, T816C, E837C, and S846C. Moreover, the remaining destabilized mutants were mainly clustered into two groups positioned from amino acid 408 to 486, and at the C terminus from amino acid 762 to 827 (Fig. 7 A, small green bars). In summary, assembly-compromised and destabilizing mutations are clustered toward the C terminus (positions 580 to 846) and between residues 408 and 486 of SbsB. Since the SDS-refractile structural motif involves the far C terminus, it can be inferred that mutations with a destabilizing effect on folding interact directly or indirectly with the C terminus. Hence, we propose that the sequence from residue 580 to 846 forms a structural domain including the C terminus. We suggest further that the sequence between amino acids 408 and 486 is either part of or interacts with the C-terminal domain. With the exception of mutants E837C and G796C, the most strongly destabilizing residues (Table I and Fig. 7 A, large green bars) are not surface accessible (Table I) and therefore most likely interact with other residues at sites buried within the C-terminal domain.
      To identify residues located at the surface of the SbsB protein, we probed the reactivity of cysteines in SbsB monomers with a hydrophilic PEG conjugate (M r 5,000). Due to its size and hydrophilicity, the reagent most likely reacts preferentially with solvent-exposed residues of the protein, while the reactivity of buried residues is slowed down or strongly diminished. The attachment of PEG to SbsB results in an increase in molecular mass, which can be readily monitored by electrophoresis. In initial studies, we used a small (M r 500) charged molecule (
      • Walker B.
      • Bayley H.
      ,
      • Krishnasastry M.
      • Walker B.
      • Braha O.
      • Bayley H.
      ), but the gel shift of the modified SbsB protein was too small to observe by SDS-PAGE. Surprisingly, in SDS-polyacrylamide gels, the PEG-SbsB conjugates migrated with an apparent molecular mass of 115–120 kDa, which is higher than the sum of the molecular masses of the protein and the polymer. A strongly decreased mobility in SDS gels was also reported for other proteins modified with PEG (
      • Kurfurst M.M.
      ,
      • Wei J.
      • Fasman G.D.
      ,
      • Wieder K.J.
      • Palczuk N.C.
      • van Es T.
      • Davis F.F.
      ). Out of 75 mutants tested 23 (31%) were very accessible to the modification and 18 (24%) moderately accessible in the monomer. The proportion of at least partly exposed residues (55%) is very high, compared with the predicted fraction (33%) of at least partly exposed residues (
      • Rost B.
      • Sander C.
      ,
      • Rost B.
      • Sander C.
      • Schneider R.
      ). The high number of surface accessible residues is, however, not surprising as 79% of the residues mutated to cysteine were polar or charged (
      • Miller S.
      • Janin J.
      • Lesk A.M.
      • Chothia C.
      ,
      • Tsai C.J.
      • Lin S.L.
      • Wolfson H.J.
      • Nussinov R.
      ).
      To identify residues positioned on the outer surface of the S-layer lattice and/or within the lumen of the pores (Fig. 1), we also probed the accessibility of the same 75 cysteines in assembled SbsB lattices, which were bound to cell wall sacculi through their inner surfaces. A PEG polymer of 5 kDa has a hydrodynamic diameter of approximately 5.2 nm assuming a spherical shape (
      • Scherrer R.
      • Gerhardt P.
      ). Given its size, it is likely, that PEG-OPSS is occluded from the pores of the S-layer lattice. Two-dimensional image-reconstruction using transmission electron micrographs of WT-SbsB lattices reveals several depressions, interpreted as pores, ranging in diameter from approximately 2.5 to 6 nm (

      Ballesteros, J. A., Deupi, X., Olivella, M., Haaksma, E. E. J., and Pardo, L. (2000) Biophys. J., in press.

      ). It is therefore likely, that PEG-OPSS is primarily reactive toward residues located at the outer surface, while residues lining the pores of the S-layer lattice are, except for the largest pores, not reactive. Residues positioned at the inner surface of the S-layer lattice (Fig. 1) are not accessible, as it is very unlikely that OPSS-PEG can permeate the cell-wall sacculi to gain access to cysteine residues. The hydrodynamic diameter of a PEG molecule of 5 kDa (5.2 nm) (
      • Scherrer R.
      • Gerhardt P.
      ) is higher than the exclusion threshold for isolated cell walls from B. megaterium (2.2 nm) (
      • Scherrer R.
      • Gerhardt P.
      ), and B. subtilis(4.2 nm) and E. coli (4.1 nm) (
      • Demchick P.
      • Koch A.L.
      ). While the determination of the thresholds was based on the uptake and permeation of test molecules (e.g. PEG and dextran) over the course of up to 2 h, it was shown that in the first 3 min after mixing test molecules the size of the cell wall pores poorly permeate the cell wall (
      • Demchick P.
      • Koch A.L.
      ). It is therefore very unlikely that OPSS-PEG penetrated the peptidoglycan meshwork during modification experiments, which were performed for a maximum of 20 s at 0 °C. Upon incorporation of SbsB monomers into the S-layer lattice, the number of accessible residues was reduced. In summary, 57 mutants were not surface accessible, six very surface accessible and 12 of intermediate surface accessibility. While the total number of very and moderately accessible residues was decreased by 56% from 41 to 18, the number of very accessible residues dropped by 74%. This drastic reduction is plausible given that (i) the inner surface of the S-layer lattice must be occluded by the cell wall and that (ii) part of the monomer surface is shielded at the subunit-subunit interface in the lattice. A decreased reactivity of cysteines might also be a result of the formation of disulfide bonds at domain-domain interfaces. However, this is not likely since the reducing agent DTT was added prior to modification with PEG-OPSS. Furthermore, no protein bands corresponding to SbsB dimers were observed upon SDS-PAGE and autoradiography of the modification mixtures. (No reducing agents were used for the SDS-PAGE.)
      Among the six very accessible residues in the lattice, five were accessible in the monomer too (T433C, S480C, S580C, D781C, and E837C), while one was of intermediate accessibility in the monomer (S651C). The increased reactivity of residue S651C upon incorporation into S-layers is significant, since it argues against the possibility that the presence of cell wall sacculi causes a general reduction in the reactivity of the protein. Residues, which were very surface accessible in the SbsB monomer were clustered into three loosely defined regions (Fig. 7 A, small red bars): cluster one, amino acid 240–281; cluster two, amino acids 433–629; cluster three, amino acids 744–913. It can be deduced that, in the native structure of SbsB, these sequences are located in regions with high surface to volume ratios. Conversely, sequences with no or very few surface accessible residues (281–433 and 629–744) might belong to globular regions with low surface to volume ratios (
      • Miller S.
      • Janin J.
      • Lesk A.M.
      • Chothia C.
      ). The high surface to volume “domains” may correspond to the low-mass spikes and bridges in the reconstructed image of Wt-SbsB (

      Ballesteros, J. A., Deupi, X., Olivella, M., Haaksma, E. E. J., and Pardo, L. (2000) Biophys. J., in press.

      ). To reinforce this interpretation, greater cysteine coverage or more independent clues would be needed.
      In summary, the examination of 75 single-cysteine mutants confirms and extends the current domain model of SbsB. Two N-terminal domains (residues 31 to 212 and 271 to 362) bind SbsB to a secondary cell wall polymer and the peptidoglycan (
      • Sára M.
      • Sleytr U.B.
      ,
      • Sára M.
      • Egelseer E.M.
      • Dekitsch C.
      • Sleytr U.B.
      ) (Fig. 7 C). In our studies, the location of the second binding domain is confirmed by the occlusion of surface accessible residues (residues 240 to 347) upon assembly of SbsB onto cell wall sacculi. The C terminus of SbsB (residues 580 to 846) forms a structural domain, which is likely to correspond to the massive morphological domain visible in computer averaged electron micrographs (

      Ballesteros, J. A., Deupi, X., Olivella, M., Haaksma, E. E. J., and Pardo, L. (2000) Biophys. J., in press.

      ). Amino residues 408 to 486 most likely interact with or are part of the C-terminal domain.

      Acknowledgement

      We thank Aida Medovich for technical assistance in the electron microscopic studies, Steve Cheley and Michael Palmer for commenting on the manuscript and Sean Conlan for assistance with preparing the cover figure.

      REFERENCES

        • Sleytr U.B.
        • Beveridge T.J.
        Trends Microbiol. 1999; 7: 253-260
        • Beveridge T.J.
        Curr. Opin. Struct. Biol. 1994; 4: 204-212
        • Sleytr U.B.
        • Messner P.
        • Pum D.
        • Sára M.
        Crystalline Bacterial Cell Surface Proteins. Landes Company/Academic Press, Austin, TX1996
        • Mesnage S.
        • Tosi-Couture E.
        • Fouet A.
        Mol. Microbiol. 1999; 31: 927-936
        • Chami M.
        • Bayan N.
        • Peyret J.L.
        • Gulik Krzywicki T.
        • Leblon G.
        • Shechter E.
        Mol. Microbiol. 1997; 23: 483-492
        • Bingle W.H.
        • Nomellini J.F.
        • Smit J.
        J. Bacteriol. 1997; 179: 601-611
        • Olabarria G.
        • Carrascosa J.L.
        • de Pedro M.A.
        • Berenguer J.
        J. Bacteriol. 1996; 178: 4765-4772
        • Dworkin J.
        • Tummuru M.K.
        • Blaser M.J.
        J. Bacteriol. 1995; 177: 1734-1741
        • Thomas S.
        • Austin J.W.
        • McCubbin W.D.
        • Kay C.M.
        • Trust T.J.
        J. Mol. Biol. 1992; 228: 652-661
        • Sára M.
        • Sleytr U.B.
        J. Bacteriol. 2000; 182: 859-868
        • Engelhardt H.
        • Peters J.
        J. Struct. Biol. 1998; 124: 276-302
        • Boot H.J.
        • Pouwels P.H.
        Mol. Microbiol. 1996; 21: 1117-1123
        • Chu S.
        • Cavaignac S.
        • Feutrier J.
        • Phipps B.M.
        • Kostrzynska M.
        • Kay W.W.
        • Trust T.J.
        J. Biol. Chem. 1991; 266: 15258-15265
        • Lupas A.
        • Engelhardt H.
        • Peters J.
        • Santarius U.
        • Volker S.
        • Baumeister W.
        J. Bacteriol. 1994; 176: 1224-1233
        • Doig P.
        • McCubbin W.D.
        • Kay C.M.
        • Trust T.J.
        J. Mol. Biol. 1993; 233: 753-765
        • Bingle W.H.
        • Nomellini J.F.
        • Smit J.
        Mol. Microbiol. 1997; 26: 277-288
        • Altenbach C.
        • Marti T.
        • Khorana H.G.
        • Hubbell W.L.
        Science. 1990; 248: 1088-1092
        • Akabas M.H.
        • Stauffer D.A.
        • Xu M.
        • Karlin A.
        Science. 1992; 258: 307-310
        • Walker B.
        • Bayley H.
        J. Biol. Chem. 1995; 270: 23065-23071
        • Frillingos S.
        • Sahin-Toth M.
        • Wu J.
        • Kaback H.R.
        FASEB J. 1998; 12: 1281-1299
        • Krishnasastry M.
        • Walker B.
        • Braha O.
        • Bayley H.
        FEBS Lett. 1994; 356: 66-71
        • Sára M.
        • Sleytr U.B.
        J. Bacteriol. 1994; 176: 7182-7189
        • Kuen B.
        • Koch A.
        • Asenbauer E.
        • Sára M.
        • Lubitz W.
        J. Bacteriol. 1997; 179: 1664-1670
        • Ries W.
        • Hotzy C.
        • Schocher I.
        • Sleytr U.B.
        • Sára M.
        J. Bacteriol. 1997; 179: 3892-3898
        • Sára M.
        • Egelseer E.M.
        • Dekitsch C.
        • Sleytr U.B.
        J. Bacteriol. 1998; 180: 6780-6783
      1. Ballesteros, J. A., Deupi, X., Olivella, M., Haaksma, E. E. J., and Pardo, L. (2000) Biophys. J., in press.

        • Sára M.
        • Kuen B.
        • Mayer H.F.
        • Mandl F.
        • Schuster K.C.
        • Sleytr U.B.
        J. Bacteriol. 1996; 178: 2108-2117
        • Miller S.
        • Janin J.
        • Lesk A.M.
        • Chothia C.
        J. Mol. Biol. 1987; 196: 641-656
        • Tsai C.J.
        • Lin S.L.
        • Wolfson H.J.
        • Nussinov R.
        Protein Sci. 1997; 6: 53-64
        • Howorka S.
        • Bayley H.
        BioTechniques. 1998; 25: 764-772
        • Jones D.H.
        Dieffenbach C.W. Dveksler G.S. PCR Primer: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1995: 591-601
        • Chung C.T.
        • Niemela S.L.
        • Miller R.H.
        Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 2172-2175
        • Huff J.P.
        • Grant B.J.
        • Penning C.A.
        • Sullivan K.F.
        BioTechniques. 1990; 9: 570-572
        • Sambrook J.
        • Fritsch E.F.
        • Maniatis T.
        Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989
        • Pum D.
        • Sára M.
        • Sleytr U.B.
        J. Bacteriol. 1989; 171: 5296-5303
        • Messner P.
        • Hollaus F.
        • Sleytr U.B.
        Int. J. Syst. Bacteriol. 1984; 34: 202-210
        • Cheley S.
        • Malghani M.S.
        • Song L.
        • Hobaugh M.
        • Gouaux J.E.
        • Yang J.
        • Bayley H.
        Protein Eng. 1997; 10: 1433-1443
        • Nakamura K.
        • Mizushima S.
        J. Biochem. 1976; 80: 1411-1422
        • Klose M.
        • Schwarz H.
        • MacIntyre S.
        • Freudl R.
        • Eschbach M.L.
        • Henning U.
        J. Biol. Chem. 1988; 263: 13291-13296
        • Kleinschmidt J.H.
        • Wiener M.C.
        • Tamm L.K.
        Protein Sci. 1999; 8: 2065-2071
        • Kurfurst M.M.
        Anal. Biochem. 1992; 200: 244-248
        • Wei J.
        • Fasman G.D.
        Biochemistry. 1995; 34: 6408-6415
        • Wieder K.J.
        • Palczuk N.C.
        • van Es T.
        • Davis F.F.
        J. Biol. Chem. 1979; 254: 12579-12587
        • Rost B.
        • Sander C.
        Proteins. 1994; 20: 216-226
        • Rost B.
        • Sander C.
        • Schneider R.
        Comp. Appl. Biosci. 1994; 10: 53-60
        • Scherrer R.
        • Gerhardt P.
        J. Bacteriol. 1971; 107: 718-735
        • Demchick P.
        • Koch A.L.
        J. Bacteriol. 1996; 178: 768-773