Salmonella enteritidis fimbriae displaying a heterologous epitope reveal a uniquely flexible structure and assembly mechanism

Two distinct Salmonella fimbrins, AgfA and SefA, comprising thin aggregative fimbriae SEF17 and SEF14, respectively, were each genetically engineered to carry PT3, an alpha-helical 16-amino acid Leishmania T-cell epitope derived from the metalloprotease gp63. To identify regions within AgfA and SefA fimbrins amenable to replacement with this epitope, PCR-generated chimeric fimbrin genes were constructed and used to replace the native chromosomal agfA and sefA genes in Salmonella enteritidis. Immunoblot analysis using anti-SEF17 and anti-PT3 sera demonstrated that all ten AgfA chimeric fimbrin proteins were expressed by S. enteritidis under normal growth conditions. Immunoelectron microscopy confirmed that eight of the AgfA::PT3 proteins were effectively assembled into cell surface-exposed fimbriae. The PT3 replacements in AgfA altered Congo red (CR) binding, cell-cell adhesion and cell surface properties of S. enteritidis to varying degrees. However, these chimeric fimbriae were still highly stable, being resistant to proteinase K digestion and requiring harsh formic acid treatment for depolymerization. In marked contrast to AgfA, none of the chimeric SefA proteins were expressed or assembled into fimbriae. Since each PT3 replacement constituted over 10% of the AgfA amino acid sequence and all ten replacements collectively represented greater than 75% of the entire AgfA primary sequence, the ability of AgfA to accept large sequence substitutions and still assemble into fibers is unique among fimbriae and other structural proteins. This structural flexibility may be related to the novel fivefold repeating sequence of AgfA and its recently proposed structure Proper formation of chimeric fimbrial fibers suggests an unusual assembly mechanism for thin aggregative fimbriae which tolerates aberrant structures. This study opens a range of possibilities for Salmonella thin aggregative fimbriae as a carrier of heterologous epitopes and as an experimental model for studies of protein structure.     

Structure and characterization of AgfB from Salmonella enteritidis thin aggregative fimbriae

The agfBAC operon of Salmonella enteritidis encodes thin aggregative fimbriae, fibrous, polymeric structures primarily composed of AgfA fimbrins. Although uncharacterized, AgfB shows a 51 % overall amino acid sequence similarity to AgfA. Using AgfB epitope-specific antiserum, AgfB was detected as a minor component of whole, purified fimbriae. Like AgfA, AgfB was released from purified fimbriae by >70 % formic acid, whereupon both AgfA-AgfA and AgfA-AgfB dimers as well as monomers were detected. This suggested that AgfB may form specific, highly stable, structural associations with AgfA in native fimbrial filaments, associations that were weakened in structurally unstable fibers derived from AgfA chimeric fimbrial mutants. Detailed sequence comparisons between AgfA and AgfB showed that AgfB harbored a similar fivefold repeated sequence pattern (x(6)QxGx(2)NxAx(3)Q), and contained structural motifs similar to the parallel beta helix model proposed for AgfA. Molecular modeling of AgfB revealed a 3D structure remarkably similar to that of AgfA, the structures differing principally in the surface disposition of non-conserved, basic, acidic and non-polar residues. Thus AgfB is a fimbrin-like structural homologue of AgfA and an integral, minor component of native thin aggregative fimbrial fibers. AgfB from an agfA deletion strain was detected as a non-fimbrial, SDS-insoluble form in the supernatant and was purified. AgfA from an agfB deletion strain was found in both SDS-soluble and insoluble, non-fimbrial forms. No AgfA-AgfA dimers were detected in the absence of AgfB. Fimbriae formation by intercellular complementation between agfB and agfA deletion strains could not be shown under a variety of conditions, indicating that AgfA and AgfB are not freely diffusible in S. enteritidis. This has important implications on the current assembly hypothesis for thin aggregative fimbriae.     

Purification and characterization of fimbriae from Salmonella enteritidis.

A human isolate of Salmonella enteritidis which displayed strong pellicle formation during static broth culture and mannose-sensitive hemagglutination produced fimbriae which were morphologically indistinguishable from type 1 fimbriae of members of the family Enterobacteriaceae. Fimbrin was purified to homogeneity, and the apparent molecular weight (Mr, 14,400) was markedly lower than that reported for the type 1 fimbrin of Salmonella typhimurium (Mr, 22,100). This fimbrin contained 40% hydrophobic amino acids and lacked cysteine. The sequence of the N-terminal 64 amino acids was determined, and sequence alignment revealed that although the 18 N-terminal residues of the S. enteritidis molecule shared considerable homology with Escherichia coli and S. typhimurium type 1 fimbrins, the S. enteritidis fimbrin lacked a 6- to 9-residue terminal sequence present in the other type 1 fimbrins and, after residue 18, shared little homology with the E. coli sequence. Antibodies raised to the purified S. enteritidis fimbrin bound to surface-exposed conformational epitopes on the native fimbriae and displayed pronounced serospecificity. These antibodies were used in the isolation of a nonfimbriated Tn10 insertion mutant which was unable to hemagglutinate.     

Prediction of secondary structure by evolutionary comparison: application to the alpha subunit of tryptophan synthase

The amino acid sequences of the a subunits of tryptophan synthase from ten different microorganisms were aligned by standard procedures. The alpha helices, beta strands and turns of each sequence were predicted separately by two standard prediction algorithms and averaged at homologous sequence positions. Additional evidence for conserved secondary structure was derived from profiles of average hydropathy and chain flexibility values, leading to a joint prediction. There is good agreement between (1) predicted beta strands, maximal hydropathy and minimal flexibility, and (2) predicted loops, great chain flexibility, and protein segments that accept insertions of various lengths in individual sequences. The a subunit is predicted to have eight repeated beta-loop-alpha-loop motifs with an extra N-terminal alpha helix and an intercalated segment of highly conserved residues. This pattern suggests that the territory structure of the a subunit is an eightfold alpha/beta barrel. The distribution of conserved amino acid residues and published data on limited proteolysis, chemical modification, and mutagenesis are consistent with the alpha/beta barrel structure. Both the active site of the a subunit and the combining site for the beta 2 subunit are at the end of the barrel formed by the carboxyl-termini of the beta strands.     

Type 1 fimbriae of Salmonella enteritidis.

Salmonella enteritidis was previously shown to produce fimbriae composed of 14,000-molecular-weight (Mr) fimbrin monomers (J. Feutrier, W. W. Kay, and T. J. Trust, J. Bacteriol. 168:221-227, 1986). Another distinct fimbrial structure, comprising 21,000-Mr fimbrin monomers, has now been identified. These fimbriae are simply designated as SEF 14 and SEF 21, respectively (for S. enteritidis fimbriae and the Mr [in thousands] of the fimbrin monomer). A simple method for the purification of both structures was developed by using the different biochemical properties of these fimbriae. SEF 21 remained intact after being boiled in sodium dodecyl sulfate but readily dissociated into subunits of 21,000 Mr at pH 2.2. The overall amino acid composition and the N-terminal amino acid sequence of the SEF 21 fimbrin were distinct from those of SEF 14 but were virtually identical to the predicted sequence for type 1 fimbrin of Salmonella typhimurium. Immunoelectron microscopy of S. enteritidis clearly revealed fimbrial structures that reacted with immune serum specific to the 21,000-Mr fimbrin. Immune sera raised against this subunit were cross-reactive with type 1 fimbrins found in whole-cell lysates of S. typhimurium, Salmonella illinois, and Salmonella cubana. However, there was no cross-reaction with Escherichia coli type 1 fimbriae or with other fimbrins produced by S. enteritidis. Under certain growth conditions, S. enteritidis produced both SEF 14 and SEF 21. However, when S. enteritidis was grown at 30 degrees C or lower, only the 21,000-Mr SEF 21 fimbrin could be detected. There was a direct correlation between mannose-sensitive hemagglutination and the presence of SEF 21.     

Analysis of the Salmonella fim gene cluster: Identification of a new gene (fimI) encoding a fimbrin-like protein and located downstream from the fimA gene

Abstract The fimA gene coding for the major component (fimbrin) of type 1 fimbriae was mapped within the Salmonella typhi fim gene cluster, and its nucleotide sequence determined. The deduced amino acid sequence of S. typhi fimbrin is highly homologous to that of S. typhimurium type 1 fimbrin and showed similarity to that of other enterobacterial type 1 fimbrins. Downstream of fimA , an open reading frame was found, named fimI , able to encode a fimbrin-like protein. The fimI product could represent the counterpart, in type 1 fimbriae, of the PapH protein involved in cell anchoring and length modulation of Escherichia coli Pap pili. This genetic organization was found to be common to other Salmonella serovars, including S. typhimurium and S. choleraesuis .     

NMR solution structure of the archaebacterial chromosomal protein MC1 reveals a new protein fold

The three-dimensional structure of methanogen chromosomal protein 1 (MC1), a chromosomal protein extracted from the archaebacterium Methanosarcina sp. CHTI55, has been solved using (1)H NMR spectroscopy. The small basic protein MC1 contains 93 amino acids (24 basic residues against 12 acidic residues). The main elements of secondary structures are an alpha helix and five beta strands, arranged as two antiparallel beta sheets (a double one and a triple one) packed in an orthogonal manner forming a barrel. The protein displays a largely hydrophilic surface and a very compact hydrophobic core made up by side chains at the interface of the two beta sheets and the helix side facing the interior of the protein. The MC1 solution structure shows a globular protein with overall dimensions in the range of 34-40 A, which potentially corresponds to a DNA-binding site of 10-12 base pairs. The presumed DNA-binding site is located on the sequence comprising residues K62-P82, which is formed by a part of strands II2 and II3 belonging to the triple-stranded antiparallel beta sheet and a loop flanked by prolines P68 and P76. The tryptophan W74 that is expected to play a key role in the DNA-binding according to photocross-linking experiments was found completely exposed to the solvent, in a good position to interact with DNA. The overall fold of MC1, characterized by its linking beta-beta-alpha-beta-beta-loop-beta, is different from other known DNA-binding proteins. Its structure suggests a different DNA-binding mode than those of the histone-like proteins HU or HMGB. Thus, MC1 may be classified as a member of a new family.     

Refined structure of spinach glycolate oxidase at 2 A resolution

The amino acid sequence of glycolate oxidase from spinach has been fitted to an electron density map of 2.0 A nominal resolution and the structure has been refined using the restrained parameter least-squares refinement of Hendrickson and Konnert. A final crystallographic R-factor of 18.9% was obtained for 32,888 independent reflections from 5.5 to 2 A resolution. The geometry of the model, consisting of 350 amino acid residues, the cofactor flavin mononucleotide and 298 solvent molecules, is close to ideal with root-mean-square deviations of 0.015 A in bond lengths and 2.6 degrees in bond angles. The expected trimodal distribution with preference for staggered conformation is obtained for the side-chain chi 1-angles. The core of the subunit is built up from the eight beta-strands in the beta/alpha-barrel. This core consists of two hydrophobic layers. One in the center is made up of residues pointing in from the beta-strands towards the barrel axis and the second, consisting of two segments of residues, pointing out from the beta-strands towards the eight alpha-helices of the barrel and pointing from the helices towards the strands. The hydrogen bond pattern for the beta-strands in the beta/alpha-barrel is described. There are a number of residues with 3(10)-helix conformation, in particular there is one left-handed helix. The ordered solvent molecules are organized mainly in clusters. The average isotropic temperature factor is quite high, 27.1 A2, perhaps a reflection of the high solvent content in the crystal. The octameric glycolate oxidase molecule, which has 422 symmetry, makes strong interactions around the 4-fold axis forming a tight tetramer, but only weak interactions between the two tetramers forming the octamer.     

Crystallographic structure of the amino terminal domain of yeast initiation factor 4A, a representative DEAD-box RNA helicase

The eukaryotic translation initiation factor 4A (elF4A) is a representative of the DEAD-box RNA helicase protein family. We have solved the crystallographic structure of the amino-terminal domain (residues 1-223) of yeast elF4A. The domain is built around a core scaffold, a parallel alpha-beta motif with five beta strands, that is found in other RNA and DNA helicases, as well as in the RecA protein. The amino acid sequence motifs that are conserved within the helicase family are localized to the beta strand-->alpha helix junctions within the core. The core of the amino terminal domain of elF4A is amplified with additional structural elements that differ from those of other helicases. The phosphate binding loop (the Walker A motif) is in an unusual closed conformation. The crystallographic structure reveals specific interactions between amino acid residues of the phosphate binding loop, the DEAD motif, and the SAT motif, whose alteration is known to impair coupling between the ATPase cycle and the RNA unwinding activity of elF4A.     

A consensus prediction of the secondary structure for the 6-phospho-β-D-galactosidase superfamily

Two separate unrefined models for the secondary structure of two subfamilies of the 6-phospho-β-D -galactosidase superfamily were independently constructed by examining patterns of variation and conservation within homologous protein sequences, assigning surface, interior, parsing, and active site residues to positions in the alignment, and identifying periodicities in these. A consensus model for the secondary structure of the entire superfamily was then built. The prediction tests the limits of an unrefined prediction made using this approach in a large protein with substantial functional and sequence divergence within the family. The protein belongs to the (α–β class), with the core β strands aligned parallel. The supersecondary structural elements that are readily identified in this model is a parallel β sheet built by strands C, D, and E, with helices 2 and 3 connecting strands (C + D) and (D + E), respectively, and an analogous α–β unit (strand G and helix 7) toward the end of the sequence. The resemblance of the supersecondary model to the tertiary structure formed by 8-fold α–β barrel proteins is almost certainly not coincidental. © 1995 Wiley-Liss, Inc.     

The structure of yeast enolase at 2.25-A resolution. An 8-fold beta + alpha-barrel with a novel beta beta alpha alpha (beta alpha)6 topology

The three-dimensional structure of yeast enolase has been determined by the multiple isomorphous replacement method followed by the solvent flattening technique. A polypeptide model, corresponding with the known amino acid sequence, has been fitted to the electron density map. Crystallographic restrained least-squares refinement of the model without solvent gave R = 20.0% for 6-2.25-A resolution with good geometry. A model with 182 water molecules and 1 sulfate which is still being refined has presently R = 17.0%. The molecule is a dimer with subunits related by 2-fold crystallographic symmetry. The subunit has dimensions 60 X 55 X 45 A and is built from two domains. The smaller N-terminal domain has an alpha + beta structure based on a three-stranded antiparallel meander and four helices. The main domain is an 8-fold beta + alpha-barrel. The enolase barrel is, however, different from the triose phosphate isomerase barrel; its topology is beta beta alpha alpha (beta alpha)6 rather than (beta alpha)8 as found in triose phosphate isomerase. The inner beta-barrel is not entirely parallel, the second strand is antiparallel to the other strands, and the direction of the first helix is also reversed with respect to the other helices. This supports the hypothesis that some enzymes evolved independently producing the stable structure of beta alpha barrels with either enolase or triose phosphate isomerase topology. The active site of enolase is located at the carboxylic end of the barrel. A fragment of the N-terminal domain and two long loops protruding from the barrel domain form a wide crevice leading to the active site region. Asp246, Glu295, and Asp320 are the ligands of the conformational cation. Other residues in the active site region are Glu168, Asp321, Lys345, and Lys396.     

Recurrent alpha beta loop structures in TIM barrel motifs show a distinct pattern of conserved structural features.

A systematic survey of seven parallel alpha/beta barrel protein domains, based on exhaustive structural comparisons, reveals that a sizable proportion of the alpha beta loops in these proteins--20 out of a total of 49--belong to either one of two loop types previously described by Thornton and co-workers. Six loops are of the alpha beta 1 type, with one residue between the alpha-helix and beta-strand, and 13 are of the alpha beta 3 type, with three residues between the helix and the strand. Protein fragments embedding the identified loops, and termed alpha beta connections since they contain parts of the flanking helix and strand, have been analyzed in detail revealing that each type of connection has a distinct set of conserved structural features. The orientation of the beta-strand relative to the helix and loop portions is different owing to a very localized difference in backbone conformation. In alpha beta 1 connections, the chain enters the beta-strand via a residue adopting an extended conformation, while in alpha beta 3 it does so via a residue in a near alpha-helical conformation. Other conserved structural features include distinct patterns of side chain orientation relative to the beta-sheet surface and of main chain H-bonds in the loop and the beta-strand moieties. Significant differences also occur in packing interactions of conserved hydrophobic residues situated in the last turn of the helix. Yet the alpha-helix surface of both types of connections adopts similar orientations relative to the barrel sheet surface. Our results suggest furthermore that conserved hydrophobic residues along the sequence of the connections, may be correlated more with specific patterns of interactions made with neighboring helices and sheet strands than with helix/strand packing within the connection itself. A number of intriguing observations are also made on the distribution of the identified alpha beta 1 and alpha beta 3 loops within the alpha/beta-barrel motifs. They often occur adjacent to each other; alpha beta 3 loops invariably involve even numbered beta-strands, while alpha beta 1 loops involve preferentially odd beta-strands; all the analyzed proteins contain at least one alpha beta 3 loop in the first half of the eightfold alpha/beta barrel. Possible origins of all these observations, and their relevance to the stability and folding of parallel alpha/beta barrel motifs are discussed.     

Purification and characterization of thin, aggregative fimbriae from Salmonella enteritidis.

Novel fimbriae were isolated and purified from the human enteropathogen Salmonella enteritidis 27655. These fimbriae were thin (measuring 3 to 4 nm in diameter), were extremely aggregative, and remained cell associated despite attempts to separate them from blended cells by centrifugation. The thin fimbriae were not solubilized in 5 M NaOH or in boiling 0.5% deoxycholate, 8 M urea, or 1 to 2% sodium dodecyl sulfate (SDS) with or without 5% beta-mercaptoethanol. Therefore, an unconventional purification procedure based on the removal of contaminating cell macromolecules in sonicated cell extracts by enzymatic digestion and preparative SDS-polyacrylamide gel electrophoresis (PAGE) was used. The insoluble fimbriae recovered from the well of the gel required depolymerization in formic acid prior to analysis by SDS-PAGE. Acid depolymerization revealed that the fimbriae were composed of fimbrin subunits, each with an apparent molecular mass of 17 kDa. Although their biochemical characteristics and amino acid composition were typical of fimbriae in general, these thin fimbriae were clearly distinct from other previously characterized fimbriae. Moreover, their fimbrin subunits had a unique N-terminal amino acid sequence. Native fimbriae on whole cells were specifically labeled with immune serum raised to the purified fimbriae. This immune serum also reacted with the denatured 17-kDa fimbrin protein in Western blots. The polyclonal immune serum did not cross-react with the other two native fimbrial types produced by this strain or with their respective fimbrins on Western blots (immunoblots). Therefore, these fimbriae represent the third fimbrial type produced by the enteropathogen S. enteritidis.     

Structural predictions for the central domain of dystrophin

The amino acid sequence of dystrophin indicates that the molecule has globular N- and C-terminal domains separated by a long central rod domain. The central rod contains multiple repeats, about 100 amino acids long and of variable length. These diverge sufficiently in sequence that, in previous studies, only 14 of the most similar repeats have been aligned and analysed in any detail. We show here that a heptad pattern of hydrophobic residues is preserved across all repeats. Using the heptad pattern together with a consensus sequence template, we identified and aligned 25 repeats in the dystrophin rod sequence. Each repeat consists of a constant-length core helix of 54 residues, coupled via a short linker to a weakly conserved variable-length helix, and then via a second linker to the next core. The variable-length helix appears truncated in repeats 10 and 13 and extended in repeats 4 and 20. The extension of repeat 20 is particularly interesting since it corresponds to a hotspot of dystrophy-inducing mutations. Detailed modelling suggests that the classical Speicher-Marchesi [(1984) Nature 311, 177-180] model for spectrin may not be appropriate to dystrophin without some modification. We propose that whilst the repeating structural motif in dystrophin is probably a bead of triple coiled coil, this bead is twice as massive as, and out of phase with, those proposed for spectrin. Our model raises the possibility that the rod domain of dystrophin may confer elasticity on the molecule. Deletions which truncate this region would then reduce the extensibility of the molecule without affecting actin crosslinking, consistent with their typically producing the relatively benign Becker phenotype of muscular dystrophy.     

Structural principles of alpha/beta barrel proteins: the packing of the interior of the sheet

Alpha/beta barrel structures very similar to that first observed in triose phosphate isomerase are now known to occur in 14 enzymes. To understand the origin of this fold, we analyzed in three of these proteins the geometry of the eight-stranded beta-sheets and the packing of the residues at the center of the barrel. The packing in this region is seen in its simplest form in glycolate oxidase. It consists of 12 residues arranged in three layers. Each layer contains four side chains. The packing of RubisCO and TIM can be understood in terms of distortions of this simple pattern, caused by residues with small side chains at some of the positions inside the barrel. Two classes of packing are found. In one class, to which RubisCO and TIM belong, the central layer is formed by a residue from the first, third, fifth, and seventh strands; the upper and lower layers are formed by residues from the second, fourth, sixth, and eighth strands. In the second class, to which GAO belongs, this is reversed: it is side chains from the even-numbered strands that form the central layer, and side chains from the odd-numbered strands that form the outer layers. Our results suggest that not all proteins with this fold are related by evolution, but that they represent a common favorable solution to the structural problems involved in the creation of a closed beta barrel.     

Structural analysis of homologous repeated domains in alpha-actinin and spectrin

The amino acid sequences of chick and slime mould alpha-actinin each contain four repeats of approximately 122 residues. These repeats are homologous to the 18-22 repeats, each of approximately 106 residues, found in the alpha and beta subunits of spectrin and fodrin, and to the multiple repeats of approximately 110 residues found in the Duchenne muscular dystrophy protein (dystrophin). The repeats correspond to the elongated rod-like portion of these molecules. We present a multiple sequence alignment of 21 repeats from this superfamily (8 alpha-actinin and 13 spectrin/fodrin), based on optimal pairwise alignments, from which a characteristic consensus pattern of amino acid types is deduced. Trp 46 is invariant in all but one repeat, and physicochemical classes of amino acids are conserved at 25 other positions. Secondary structure prediction on both the alpha-actinin and spectrin repeats taken together with the distribution of proline residues in the sequences, strongly suggest that each repeated domain consists of a four-helix structure. Our predictions differ significantly from previous three-helix models based on analyses of fewer sequences. To determine possible interdomain regions, sites of limited proteolysis of the native chick alpha-actinin dimer were determined and located in the amino acid sequence. The majority of these sites were in corresponding positions in different repeats within a segment predicted as a long helix. We propose a model, consistent with the overall dimensions of the rod-like portions of the molecules, in which these long, probably interrupted helices, link adjacent domains.     

TMBHMM: a frequency profile based HMM for predicting the topology of transmembrane beta barrel proteins and the exposure status of transmembrane residues

Transmembrane beta barrel (TMB) proteins are found in the outer membranes of bacteria, mitochondria and chloroplasts. TMBs are involved in a variety of functions such as mediating flux of metabolites and active transport of siderophores, enzymes and structural proteins, and in the translocation across or insertion into membranes. We present here TMBHMM, a computational method based on a hidden Markov model for predicting the structural topology of putative TMBs from sequence. In addition to predicting transmembrane strands, TMBHMM also predicts the exposure status (i.e., exposed to the membrane or hidden in the protein structure) of the residues in the transmembrane region, which is a novel feature of the TMBHMM method. Furthermore, TMBHMM can also predict the membrane residues that are not part of beta barrel forming strands. The training of the TMBHMM was performed on a non-redundant data set of 19 TMBs. The self-consistency test yielded Q(2) accuracy of 0.87, Q(3) accuracy of 0.83, Matthews correlation coefficient of 0.74 and SOV for beta strand of 0.95. In this self-consistency test the method predicted 83% of transmembrane residues with correct exposure status. On an unseen, non-redundant test data set of 10 proteins, the 2-state and 3-state TMBHMM prediction accuracies are around 73% and 72%, respectively, and are comparable to other methods from the literature. The TMBHMM web server takes an amino acid sequence or a multiple sequence alignment as an input and predicts the exposure status and the structural topology as output. The TMBHMM web server is available under the tmbhmm tab at: http://service.bioinformatik.uni-saarland.de/tmx-site/.     

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.