X-ray structure determination of telokin, the C-terminal domain of myosin light chain kinase, at 2.8 A resolution.

The three-dimensional structure of telokin, an acidic protein identical to the C-terminal portion of smooth muscle myosin light chain kinase from turkey gizzard, has been determined at 2.8 A resolution and refined to a crystallographic R-factor of 19.5% for all measured X-ray data from 30 A to 2.8 A. Crystals used in the investigation belonged to the space group P3(2)21, with one molecule per asymmetric unit and unit cell dimensions of a = b = 64.4 A and c = 50.6 A. Telokin contains 154 amino acid residues, 103 of which were visible in the electron density map. The overall molecular fold of telokin consists of seven strands of antiparallel beta-pleated sheet that wrap around to form a barrel. There is also an extended tail of eight amino acid residues at the N terminus that does not participate in beta-sheet formation. The beta-barrel can be simply envisioned as two layers of beta-sheet, nearly parallel to one another, with one layer containing four and the other three beta-strands. This type of beta-barrel, as seen in telokin, was first observed for the CH2 domain of an immunoglobulin fragment Fc. Telokin is an intracellular protein and, as such, does not contain the disulphide linkage between beta-strands B and F normally observed in the immunoglobulin constant domains. It does, however, contain two cysteine amino acid residues (Cys63 and Cys115) that are situated at structurally identical positions to those forming the disulphide linkage in the immunoglobulin constant domain.     

Quantitative evaluation of congo red binding to amyloid-like proteins with a beta-pleated sheet conformation

The binding of Congo red to several purified amyloid-like peptides having a beta-pleated sheet conformation was quantitatively examined. Congo red binds preferentially to the beta-pleated sheet conformation of both insulin fibrils and poly-L-lysine. Congo red does not bind nearly so well to poly-L-serine or polyglycine, despite the fact that these peptides also have a beta-pleated sheet conformation. Binding to insulin fibrils was saturable with an apparent Bmax of 2 moles of Congo red per mole of insulin fibrils and an apparent KD of 1.75 x 10(-7) M. Binding to beta-poly-L-lysine was similar but had a much higher apparent Bmax of 43. Binding of Congo red to beta-poly-L-lysine was pH dependent and appeared to be determined by the number of protonated lysine residues in the 250 amino acid peptide. We present a new hypothesis in which Congo red binds to amyloid-like proteins via bonds between the two negatively charged sulfonic acid groups of Congo red and two positively charged amino acid residues of two separate protein molecules which are properly oriented by virtue of the beta-pleated sheet conformation of the peptide backbone.     

The molecular structure of UDP-galactose 4-epimerase from Escherichia coli determined at 2.5 A resolution.

UDP-galactose 4-epimerase catalyzes the conversion of UDP-galactose to UDP-glucose during normal galactose metabolism. The molecular structure of UDP-galactose 4-epimerase from Escherichia coli has now been solved to a nominal resolution of 2.5 A. As isolated from E. coli, the molecule is a dimer of chemically identical subunits with a total molecular weight of 79,000. Crystals of the enzyme used for this investigation were grown as a complex with the substrate analogue, UDP-benzene, and belonged to the space group P2(1)2(1)2(1) with unit cell dimensions of a = 76.3 A, b = 83.1 A, c = 132.1 A, and one dimer per asymmetric unit. An interpretable electron density map calculated to 2.5 A resolution was obtained by a combination of multiple isomorphous replacement with six heavy atom derivatives, molecular averaging, and solvent flattening. Each subunit of epimerase is divided into two domains. The larger N-terminal domain, composed of amino acid residues 1-180, shows a classic NAD+ binding motif with seven strands of parallel beta-pleated sheet flanked on either side of alpha-helices. The seventh strand of the beta-pleated sheet is contributed by amino acid residues from the smaller domain. In addition, this smaller C-terminal domain, consisting of amino acid residues 181-338, contains three strands of beta-pleated sheet, two major alpha-helices and one helical turn. The substrate analogue, UDP-benzene, binds in the cleft located between the two domains with its phenyl ring in close proximity to the nicotinamide ring of NAD+. Contrary to the extensive biochemical literature suggesting that epimerase binds only one NAD+ per functional dimer, the map clearly shows electron density for two nicotinamide cofactors binding in symmetry-related positions in the dimer. Likewise, each subunit in the dimer also binds one substrate analogue.     

Solution structure of At3g04780.1-des15, an Arabidopsis thaliana ortholog of the C-terminal domain of human thioredoxin-like protein

The structure of At3g04780.1-des15, an Arabidopsis thaliana ortholog of the C-terminal domain of human thioredoxin-like protein, was determined by NMR spectroscopy. The structure is dominated by a beta-barrel sandwich. A two-stranded anti-parallel beta-sheet, which seals off one end of the beta-barrel, is flanked by two flexible loops rich in acidic amino acids. Although this fold often provides a ligand binding site, the structure did not reveal an appreciable cavity inside the beta-barrel. The three-dimensional structure of At3g04780.1-des15 provides an entry point for understanding its functional role and those of its mammalian homologs.     

Structural and functional features of Drosophila chorion proteins s36 and s38 from analysis of primary structure and infrared spectroscopy

Amino acid composition, Fourier transform analysis and secondary structure prediction methods strongly support a tripartite structure for Drosophila chorion proteins s36 and s38. Each protein consists of a central domain and two flanking 'arms'. The central domain contains tandemly repetitive peptides, which apparently generate a secondary structure of beta-sheet strands alternating with beta-turns, most probably, forming a twisted beta-pleated sheet or beta-barrel. The central domains of s36 and s38 share similarities, but they are recognizably different. The flanking 'arms', with different primary and secondary structure features, presumably serve protein-specific functions. The possible roles of the protein domains for the establishment of higher order structure in Drosophila chorion and the possible function of the molecules are discussed. The predicted secondary structure of Drosophila chorion proteins s36 and s38 is supported by experimental information obtained from Fourier transform infrared spectroscopic studies of Drosophila chorions.     

Three-dimensional structure of the ribonuclease T1 2'-GMP complex at 1.9-A resolution

The complex formed between the enzyme ribonuclease T1 (EC 3.1.27.3) and its specific inhibitor 2'-guanylic acid (2'-GMP) has been refined to R = 0.180 using x-ray diffraction data to 1.9-A resolution. The protein molecule displays a compact fold; a 4.5 turn alpha-helix packed over an antiparallel beta-pleated sheet shields most of the hydrophobic interior of the protein against the solvent. The extended pleated sheet structure of ribonuclease T1 is composed of three long and four short strands building up a two-stranded minor beta-sheet near the amino terminus and a five-stranded major sheet in the interior of the protein molecule. In the complex with ribonuclease T1, the inhibitor 2'-guanylic acid adopts the syn-conformation and C2'-endo sugar pucker. Binding of the nucleotide is mainly achieved through amino acid residues 38-46 of the protein. The catalytically active amino acid residues of ribonuclease T1 (His40, Glu58, Arg77, and His92) are located within the major beta-sheet which, as evident from the analysis of atomic temperature factors, provides an environment of minimal local mobility. The geometry of the active site is consistent with a mechanism for phosphodiester hydrolysis where, in the transesterification step, His40 and/or Glu58 act as a general base toward the ribose 2'-hydroxyl group and His92, as a general acid, donates a proton to the leaving 5'-hydroxyl group.     

Amino acid sequence of the blue copper protein rusticyanin from Thiobacillus ferrooxidans

Rusticyanin is a small blue copper protein isolated from Thiobacillus ferrooxidans. The amino acid sequence of the rusticyanin has been determined by the structural characterization of tryptic and endoproteinase Asp-N peptides with use of amino terminal microsequencing, fast atom bombardment mass spectrometry, and electrospray triple-quadrupole mass spectrometry techniques. Amino acid analysis, carboxy-terminal sequence analysis, and circular dichroism spectroscopy were also performed on the protein. Amino acid sequence identity among rusticyanin and six other small blue copper proteins is apparent only in the limited C-terminal region of each protein bearing three of the four putative copper ligands. A structural model of the rusticyanin is proposed where the protein is principally a beta-barrel comprised of six strands. This model is consistent with the circular dichroism data and computational predictions of the secondary structure of rusticyanin. A feature of the model is the hypothesis that Asp 73 may serve as a fourth copper ligand.     

The molecular structure of insecticyanin from the tobacco hornworm Manduca sexta L. at 2.6 A resolution.

Insecticyanin, a blue biliprotein isolated from the tobacco hornworm Manduca sexta L., is involved in insect camouflage. Its three-dimensional structure has now been solved to 2.6 A resolution using the techniques of multiple isomorphous replacement, non-crystallographic symmetry averaging about a local 2-fold rotation axis and solvent flattening. All 189 amino acids have been fitted to the electron density map. The map clearly shows that insecticyanin is a tetramer with one of its molecular 2-fold axes coincident to a crystallographic dyad. The individual subunits have overall dimensions of 44 A X 37 A X 40 A and consist primarily of an eight-stranded anti-parallel beta-barrel flanked on one side by a 4.5-turn alpha-helix. Interestingly the overall three-dimensional fold of the insecticyanin subunit shows remarkable similarity to the structural motifs of bovine beta-lactoglobulin and the human serum retinol-binding protein. The electron density attributable to the chromophore is unambiguous and shows that it is indeed the gamma-isomer of biliverdin. The biliverdin lies towards the open end of the beta-barrel with its two propionate side chains pointing towards the solvent and it adopts a rather folded conformation, much like a heme.     

Turn scanning by site-directed mutagenesis: application to the protein folding problem using the intestinal fatty acid binding protein

We have systematically mutated residues located in turns between beta-strands of the intestinal fatty acid binding protein (IFABP), and a glycine in a half turn, to valine and have examined the stability, refolding rate constants and ligand dissociation constants for each mutant protein. IFABP is an almost all beta-sheet protein exhibiting a topology comprised of two five-stranded sheets surrounding a large cavity into which the fatty acid ligand binds. A glycine residue is located in seven of the eight turns between the antiparallel beta-strands and another in a half turn of a strand connecting the front and back sheets. Mutations in any of the three turns connecting the last four C-terminal strands slow the folding and decrease stability with the mutation between the last two strands slowing folding dramatically. These data suggest that interactions between the last four C-terminal strands are highly cooperative, perhaps triggered by an initial hydrophobic collapse. We suggest that this trigger is collapse of the highly hydrophobic cluster of amino acids in the D and E strands, a region previously shown to also affect the last stage of the folding process (Kim et al., 1997). Changing the glycine in the strand between the front and back sheets also results in a unstable, slow folding protein perhaps disrupting the D-E strand interactions. For most of the other turn mutations there was no apparent correlation between stability and refolding rate constants. In some turns, the interaction between strands, rather than the turn type, appears to be critical for folding while in others, turn formation itself appears to be a rate limiting step. Although there is no simple correlation between turn formation and folding kinetics, we propose that turn scanning by mutagenesis will be a useful tool for issues related to protein folding.     

Delta98delta, a functional all-beta-sheet abridged form of intestinal fatty acid binding protein

Intestinal fatty acid binding protein (IFABP) is a 15 kDa intracellular lipid-binding protein exhibiting a beta-barrel fold that resembles a clamshell. The beta-barrel, which encloses the ligand binding cavity, consists of two perpendicular five-stranded beta-sheets with an intervening helix-turn-helix motif between strands A and B. Delta98delta (fragment 29-126 of IFABP) was obtained either in its recombinant form or by limited proteolysis with clostripain. Despite lacking extensive stretches involved in the closure of the beta-barrel, delta98delta remains soluble and stable in solution. Spectroscopic analyses by circular dichroism, ultraviolet absorption, and intrinsic fluorescence indicate that the fragment retains substantial beta-sheet content and tertiary interactions. In particular, the environment around W82 is identical in both delta98delta and IFABP, a fact consistent with the conservation in the former of all the critical amino acid residues belonging to the hydrophobic core. In addition, the Stokes radius of delta98delta is similar to that of IFABP and 16% larger than that calculated from its molecular weight (11 kDa). The monomeric status of delta98delta was further confirmed by chemical cross-linking experiments. Although lacking 25% of the amino acids of the parent protein, in the presence of GdnHCl, delta98delta unfolds through a cooperative transition showing a midpoint at 0.90 M. Remarkably, it also preserves binding activity for fatty acids (Kd = 5.1 microM for oleic acid and Kd = 0.72 microM for trans-parinaric acid), a fact that exerts a stabilizing effect on its structure. These cumulative evidences show that delta98delta adopts a monomeric state with a compact core and a loose periphery, being so far the smallest structure of its kind preserving binding function.     

Identification of conserved residue patterns in small beta-barrel proteins

Our abilities to predict three-dimensional conformation of a polypeptide, given its amino acid sequence, remain limited despite advances in structure analysis. Analysis of structures and sequences of protein families with similar secondary structural elements, but varying topologies, might help in addressing this problem. We have studied the small beta-barrel class of proteins characterized by four strands (n = 4) and a shear number of 8 (S = 8) to understand the principles of barrel formation. Multiple alignments of the various protein sequences were generated for the analysis. Positional entropy, as a measure of residue conservation, indicated conservation of non-polar residues at the core positions. The presence of a type II beta-turn among the various barrel proteins considered was another strikingly invariant feature. A conserved glycyl-aspartyl dipeptide at the beta-turn appeared to be important in guiding the protein sequence into the barrel fold. Molecular dynamics simulations of the type II beta-turn peptide suggested that aspartate is a key residue in the folding of the protein sequence into the barrel. Our study suggests that the conserved type II beta-turn and the non-polar residues in the barrel core are crucial for the folding of the protein's primary sequence into the beta-barrel conformation.     

Molecular cloning, sequencing, and expression of omp-40, the gene coding for the major outer membrane protein from the acidophilic bacterium Thiobacillus ferrooxidans

Thiobacillus ferrooxidans is one of the chemolithoautotrophic bacteria important in industrial biomining operations. Some of the surface components of this microorganism are probably involved in adaptation to their acidic environment and in bacterium-mineral interactions. We have isolated and characterized omp40, the gene coding for the major outer membrane protein from T. ferrooxidans. The deduced amino acid sequence of the Omp40 protein has 382 amino acids and a calculated molecular weight of 40,095.7. Omp40 forms an oligomeric structure of about 120 kDa that dissociates into the monomer (40 kDa) by heating in the presence of sodium dodecyl sulfate. The degree of identity of Omp40 amino acid sequence to porins from enterobacteria was only 22%. Nevertheless, multiple alignments of this sequence with those from several OmpC porins showed several important features conserved in the T. ferrooxidans surface protein, such as the approximate locations of 16 transmembrane beta strands, eight loops, including a large external L3 loop, and eight turns which allowed us to propose a putative 16-stranded beta-barrel porin structure for the protein. These results together with the previously known capacity of Omp40 to form ion channels in planar lipid bilayers strongly support its role as a porin in this chemolithoautotrophic acidophilic microorganism. Some characteristics of the Omp40 protein, such as the presence of a putative L3 loop with an estimated isoelectric point of 7.21 allow us to speculate that this can be the result of an adaptation of the acidophilic T. ferrooxidans to prevent free movement of protons across its outer membrane.     

The 2 A resolution structure of the sulfate-binding protein involved in active transport in Salmonella typhimurium

The crystal structure of the liganded form of the sulfate-binding protein, an initial receptor for active transport of sulfate in Salmonella typhimurium, has been solved and refined at 2.0 A resolution (1 A = 0.1 nm). The final model, which consists of 2422 non-hydrogen atoms, one sulfate substrate and 143 water molecules, yields a crystallographic R-factor of 14.0% for 16,959 reflections between 8 and 2 A. The structure deviates from ideal bond lengths and angle distances by 0.015 A and 0.037 A, respectively. The protein is ellipsoid with overall dimensions of 35 A x 35 A x 65 A and consists of two similar globular domains. The two domains are linked by three distinct peptide segments, which though widely separated in the amino acid sequence, are in close proximity in the tertiary structure. As these connecting segments are located near the periphery of the molecule, they further serve as the base or a "boundary" of the deep cleft formed between the two domains. Despite the unusual interdomain connectivity, both domains have similar supersecondary structure consisting of a central five-stranded beta-pleated sheet sandwiched by alpha-helices on either side. The arrangement of the two domains gives rise to the ellipsoidal shape and to the cleft between the two domains wherein the sulfate substrate is found and completely engulfed. A discovery of considerable importance is that the sulfate substrate is tightly held in place primarily by seven hydrogen bonds, five of which are donated by main-chain peptide NH groups, another by a serine hydroxyl and the last by the indole NH moiety of a tryptophan side-chain; there are no positively charged residues, nor cations, nor water molecules within van der Waals' distance to the sulfate dianion. All the main-chain peptide units associated with the sulfate are in turn linked (via the peptide CO group) to arrays of hydrogen bonds. Three of these arrays are composed of alternating peptide units and hydrogen bonds within the solvent-exposed part of three alpha-helices and two are linked to a histidine and an arginine residue. The sulfate-binding protein bears strong similarity to the structures of four other periplasmic binding proteins solved in our laboratory which are specific for L-arabinose, D-galactose/D-glucose, leucine/isoleucine/valine and leucine. The similarity includes the ellipsoidal shape and the two globular domain structures, each domain consisting of a central beta-pleated sheet flanked by alpha-helices.(ABSTRACT TRUNCATED AT 400 WORDS)     

Crystal structure of a novel trimethoprim-resistant dihydrofolate reductase specified in Escherichia coli by R-plasmid R67

Crystalline R67 dihydrofolate reductase (DHFR) is a dimeric molecule with two identical 78 amino acid subunits, each folded into a beta-barrel conformation. The outer surfaces of the three longest beta strands in each protomer together form a third beta barrel having six strands at the subunit interface. A unique feature of the enzyme structure is that while the intersubunit beta barrel is quite regular over most of its surface, an 8-A "gap" runs the full length of the barrel, disrupting potential hydrogen bonds between beta-strand D in subunit I and the adjacent corresponding strand of subunit II. It is proposed that this deep groove is the NADPH binding site and that the association between protein and cofactor is modulated by hydrogen-bonding interactions along one face of this antiparallel beta-barrel structure. A hypothetical model is proposed for the R67 DHFR-NADPH-folate ternary complex that is consistent with both the known reaction stereoselectivity and the weak binding of 2,4-diamino inhibitors to the plasmid-specified reductase. Geometrical comparison of this model with an experimentally determined structure for chicken DHFR suggests that chromosomal and type II R-plasmid specified enzymes may have independently evolved similar catalytic machinery for substrate reduction.     

The amino acid sequence of low-potential cytochrome c550 from the cyanobacterium Microcystis aeruginosa

The low-potential cytochrome c550 has been purified from the cyanobacterium Microcystis aeruginosa and its amino acid sequence has been determined. The protein contains 135 amino acid residues with the Cys-X-X-Cys-His heme binding site at residues 37 to 41. The sequence from residue 28 to 45 shows similarity to cytochrome c553 residues 1 to 18 when the heme binding sites are aligned. Another region of similarity is in the carboxyl-terminal regions of these two proteins. The two aligning regions of cytochrome c553 correspond to helical segments in other related cytochromes. A partial sequence of cytochrome c550 from Aphanizomenon flos-aquae was obtained and showed a 48% identity to the sequence of the M. aeruginosa cytochrome. The single methionine residue in cytochrome c550 of M. aeruginosa occurs at position 119 but there is no methionine in this region in the A. flos-aquae cytochrome, indicating that methionine is not the sixth ligand to the heme iron atom. Histidine 92 is a possible sixth ligand in M. aeruginosa cytochrome c550. The far-uv circular dichroism spectrum indicates that this protein is approximately 17% alpha helix, 42% beta-pleated sheet, and 41% random coil.     

Specificity determinants for lipids bound to beta-barrel proteins

The family of proteins accountable for the intracellular movement of lipids is characterized by a 10-stranded beta-barrel that forms an internalized cavity varying in size and binding preferences. The loop connecting beta-strands E and F (the fifth and sixth strands) is the most striking conformational difference between adipocyte lipid binding protein (ALBP; fatty acids) and cellular retinoic acid binding protein type I (CRABP I). A three-residue mutation was made in wild-type (WT)-ALBP [ALBP with a three-residue mutation (EF-ALBP)] to mimic CRABP I. Crystal structures of ligand-free and EF-ALBP with bound oleic acid were solved to resolutions of 1.5 A and 1.7 A, respectively, and compared with previous studies of WT-ALBP. The changes in three residues of one loop of the protein appear to have altered the positioning of the C18 fatty acid, as observed in the electron density of EF-ALBP. The crystallographic studies made it possible to compare the protein conformation and ligand positioning with those found in the WT protein. Although the cavity binding sites in both the retinoid and fatty acid binding proteins are irregular, the ligand atoms appear to favor a relatively planar region of the cavities. Preliminary chemical characterization of the mutant protein indicated changes in some binding properties and overall protein stability.     

The role of DRB1 amino acid residue differences on donor-specific antibody production and acute rejection after cadaveric renal transplant.

The correlation between DRB1 amino acid residue matching, post-transplant humoral response and acute rejection (ARj) episodes was analysed in 51 renal transplant donor-recipient pairs in order to determine new criteria for organ assignment based on the alloreactivity of the residue within the peptide binding groove. HLA class I and II compatibility was defined using serological and genomic techniques; a sequence-based typing (SBT) was used for a higher resolution of DRB1 alleles. Humoral response was monitored in the first post-transplant year using triple staining flow cytometric analysis of donor-specific antibodies (Abs). Our data showed that DRB1 residue compatibility was always correlated to the absence of ARj while the presence of one or more aminoacid differences was associated with a similar frequency of ARj. Analysis of the mismatched DRB1 amino acid residue localised in the beta-pleated sheet and the alpha-helix of the DRB 1 molecule revealed that the frequency of beta-pleated sheet residue mismatches (MMs) was higher in the ARj-positive than in the ARj-negative group. A significant increase in the alpha-helix residue MMs was observed in patients with anti-class II Ab production (p = 0.034). Furthermore, analysing in detail DRB 1 MMs at the level of single amino acid residue, we found that the frequency of the mismatches localized in codon 9 and codon 28 in the beta-pleated sheet, as well as in codon 57 in the alpha-helix, was higher in patients who experienced ARj; on the other hand, MMs in codon 58 of the alpha-helix were more frequently associated with anti-class II Ab production. The identification of the residues more involved in alloreactivity onset will make it possible to define the existence of "permissive" or immunogenic" allele combinations which could simplify and increase the chances of a successful transplant.     

Structural analysis and comparison of cobrotoxin and cardiotoxins by near-IR Fourier transform Raman spectroscopy.

Venom toxins were isolated from Formosan cobra (Naja naja atra) by cation-exchange chromatography. The near-IR FT-Raman analytical method has been applied to the characterization and classification of the toxin components in their lyophilized forms. Structural analysis and comparison of various purified toxin fractions were made with respect to their amino acid compositions and near-IR Fourier-transform Raman spectra. The results indicate that the major secondary structure of cobra toxins including cobrotoxin and various cardiotoxins is mainly anti-parallel beta-pleated sheet as judged by the Raman signals at 1238 cm-1 (amide III) and 1671 cm-1 (amide I). It is also found that the relative Raman signal intensities of Tyr, Phe, Trp and Met residues in purified toxins correlate very well with the structural data obtained from amino acid analysis. The advantage and improvement of applying the near-IR FT-Raman spectroscopy to the unambiguous classification and comparison of venom toxins are evident and the discrepancies with previous Raman studies on these venom toxins are also revealed and discussed.     

The function of OmpA in Escherichia coli

Outer membrane protein A (OmpA) is a major protein in the Escherichia coli outer membrane. In this study, the function of OmpA in E. coli stress survival was examined. An E. coli K1 ompA-deletion mutant was significantly more sensitive than that of its parent strain to sodium dodecyl sulfate (SDS), cholate, acidic environment, high osmolarity, and pooled human serum. A number of amino acid changes at the extracellular loops of OmpA did not affect the viability of E. coli, while short peptide insertions in the periplasmic turns of the OmpA beta-barrel decreased E. coli resistance to environmental stresses. Moreover, ompA mutants were found to survive much better within brain microvascular endothelial cells than the wild-type strain, supporting that OmpA is a major target in mammalian host cell defense. These results indicated that OmpA plays a vital structural role in E. coli, and suggested that a perfect beta-barrel structure of OmpA is important for outer membrane stability. Based on these results and the published OmpA structural analyses, I propose that OmpA is composed of three functional domains including a hydrophilic extracellular mass, a beta-barrel transmembrane structure, and a peptidoglycan binding domain.     

Monte Carlo studies on equilibrium globular protein folding. II. Beta-barrel globular protein models

In the context of dynamic Monte Carlo simulations on a model protein confined to a tetrahedral lattice, the interplay of protein size and tertiary structure, and the requirements for an all-or-none transition to a unique native state, are investigated. Small model proteins having a primary sequence consisting of a central bend neutral region flanked by two tails having an alternating hydrophobic/hydrophilic pattern of residues are seen to undergo a continuous transition to a beta-hairpin collapsed state. On increasing the length of the tails, the beta-hairpin structural motif is found to be in equilibrium with a four-member beta-barrel. Further increase of the tail length results in the shift of the structural equilibrium to the four-member beta-barrel. The random coil to beta-barrel transition is of an all-or-none character, but while the central turn is always the desired native bend, the location of the turns involving the two external strands is variable. That is, beta-barrels having the external stands that are two residues out of register are also observed in the transition region. Introduction into the primary sequence of two additional regions that are at the very least neutral toward turn formation produces an all-or-none transition to the unique, native, four-member beta-barrel. Various factors that can augment the stability of the native conformation are explored. Overall, these folding simulations strongly indicate that the general rules of globular protein folding are rather robust--namely, one requires a general pattern of hydrophobic/hydrophilic residues that allow the protein to have a well-defined interior and exterior and the presence of regions in the amino acid sequence that at the very least are locally indifferent to turn formation. Since no site-specific interactions between hydrophobic and hydrophilic residues are required to produce a unique four-member beta-barrel, these simulations strongly suggest that site specificity is involved in structural fine-tuning.