Molecular dynamics simulations of a highly charged peptide from an SH3 domain: possible sequence-function relationship

A seven-residue peptide that is highly conserved in SH3 domains despite being far from the active site has been shown by NMR to be stable in solution. This peptide, biologically important because it is a likely folding nucleus for SH3 domains, provides a challenging subject for molecular dynamics because it is highly charged. We present stable, 10-ns simulations of both the native-like diverging turn structure and a helical model. Free energies of these two conformations, estimated through MM-PBSA analysis using several force fields, suggest a comparable free energy (DeltaDeltaG < or =6 kcal/mol) for native and helix conformations. NOE intensities calculated from the native trajectory reproduce experimental data quite well, suggesting that the conformations sampled by the trajectory reasonably represent those observed in the NMR experiment. The molecular dynamics results, as well as sequence analysis of a diverse 690-member family of SH3 domain proteins, suggest that the presence of two elements is essential for formation of the diverging turn structure: a pair of residues with low helical propensity in the turn region and, as previously recognized, two hydrophobic residues to close the end of the diverging turn. Thus, these two sequence features may form the structural basis for the function of this peptide as a folding nucleus in this family of proteins.     

Spatial separation of beta-sheet domains of beta-amyloid: disruption of each beta-sheet by N-methyl amino acids

In a recent model of beta-amyloid (Abeta) fibrils, based mainly on solid-state NMR data, a molecular layer consists of two beta-sheets (residues 12-23 and 31-40 of Abeta1-40), folded onto one another by a connecting "bend" structure (residues 25-29) in the side-chain dimension. In this paper, we use two N-methyl amino acids to disrupt each of the two beta-sheets individually (2NMe(NTerm), residues 17 and 19; and 2NMe(CTerm), residues 37 and 39), or both of them at the same time (4NMe, with the above four N-methylated residues). Our data indicate that incorporation of two N-methyl amino acids into one beta-sheet is sufficient to disrupt that sheet while leaving the other, unmodified beta-sheet intact and able to form fibrils. We show, however, that disruption of each of the two beta-sheets has strikingly different effects on fibrillogenesis kinetics and fibril morphology. Both 2NMe(NTerm) and 2NMe(CTerm) form fibrils at similar rates, but more slowly than that of unmodified Abeta1-40. Electron microscopy shows that 2NMe(NTerm) forms straight fibrils with fuzzy amorphous material coating the edges, while 2NMe(CTerm) forms very regular, highly twisted fibrils-in both cases, distinct from the morphology of Abeta1-40 fibrils. Both 2NMe peptides show a "CMC" approximately four times greater than that of Abeta1-40. CD spectra of these peptides also evolve differently in time: whereas the CD spectra of 2NMe(NTerm) evolve little over 10 days, those of 2NMe(CTerm) show a transition to high beta-sheet content at about day 4-5. We also show that disruption of both beta-sheet domains, as in 4NMe, prevents fibril formation altogether, and renders Abeta1-40 highly water soluble and monomeric, and with solvent-exposed side chains. In summary, our data show (1) that the two beta-sheet domains fold in a semiautonomous manner, since disrupting each one still allows the other to fold; (2) that disruption of the N-terminal beta-sheet has a more profound effect on fibrillogenesis than disruption of the C-terminal beta-sheet, suggesting that the former is the more critical for the overall structure of the fibril; and (3) that disruption of both beta-sheet domains renders the peptide monomeric and unable to form fibrils.     

Factors involved in the stability of isolated beta-sheets: Turn sequence, beta-sheet twisting, and hydrophobic surface burial

We have recently reported on the design of a 20-residue peptide able to form a significant population of a three-stranded up-and-down antiparallel beta-sheet in aqueous solution. To improve our beta-sheet model in terms of the folded population, we have modified the sequences of the two 2-residue turns by introducing the segment DPro-Gly, a sequence shown to lead to more rigid type II' beta-turns. The analysis of several NMR parameters, NOE data, as well as Deltadelta(CalphaH), DeltadeltaC(beta), and Deltadelta(Cbeta) values, demonstrates that the new peptide forms a beta-sheet structure in aqueous solution more stable than the original one, whereas the substitution of the DPro residues by LPro leads to a random coil peptide. This agrees with previous results on beta-hairpin-forming peptides showing the essential role of the turn sequence for beta-hairpin folding. The well-defined beta-sheet motif calculated for the new designed peptide (pair-wise RMSD for backbone atoms is 0.5 +/- 0.1 A) displays a high degree of twist. This twist likely contributes to stability, as a more hydrophobic surface is buried in the twisted beta-sheet than in a flatter one. The twist observed in the up-and-down antiparallel beta-sheet motifs of most proteins is less pronounced than in our designed peptide, except for the WW domains. The additional hydrophobic surface burial provided by beta-sheet twisting relative to a "flat" beta-sheet is probably more important for structure stability in peptides and small proteins like the WW domains than in larger proteins for which there exists a significant contribution to stability arising from their extensive hydrophobic cores.     

Solution structure of midkine, a new heparin-binding growth factor.

Midkine (MK) is a 13 kDa heparin-binding polypeptide which enhances neurite outgrowth, neuronal cell survival and plasminogen activator activity. MK is structurally divided into two domains, and most of the biological activities are located on the C-terminal domain. The solution structures of the two domains were determined by NMR. Both domains consist of three antiparallel beta-strands, but the C-terminal domain has a long flexible hairpin loop where a heparin-binding consensus sequence is located. Basic residues on the beta-sheet of the C-terminal domain form another heparin-binding site. Measurement of NMR signals in the presence of a heparin oligosaccharides verified that multiple amino acids in the two sites participated in heparin binding. The MK dimer has been shown to be the active form, giving signals to endothelial cells and probably to neuronal cells. We present a head-to-head dimer model of MK. The model was supported by the results of cross-linking experiments using transglutaminase. The dimer has a fused heparin-binding site at the dimer interface of the C-terminal domain, and the heparin-binding sites on MK fit the sulfate group clusters on heparin. These features are consistent with the proposed stronger heparin-binding activity and biological activity of the dimer.     

Structural transitions involved in a novel amyloid-like beta-sheet assemblage of tripeptide derivatives

Self-assembly of two tripeptide derivatives containing three nonpolar isoleucine moieties and polar oxyethylene groups are studied in methanol. Peptide A [CH3(OCH2CH2)3OCH2CO(Ile)3OCH3] and peptide B [CH3(OCH2CH2)3OCH2CO(Ile)3NH (CH2CH2O)3CH3] take a mixture of unordered and helical conformation at low concentration (8.5 x 10(-4) M). However, at high concentration (2 x 10(-3) M), both the peptide showed significant increase in the helical conformation. An interesting conformational transition of peptides A and B at various methanol contents was observed in the solvated films of these compounds by spectroscopic methods like the far-uv circular dichroism and Fourier transform infrared (FT-IR) techniques. Peptide B, which contains more polar oxyethylene groups than A, showed a highly cooperative conformational transition when the methanol content was decreased. This transition was characterized by a large increase of beta-sheet, retaining a alpha-helical contribution. Peptide A showed a conformational transition resulting in a beta-sheet in the aggregated state. From the CD spectra, the ratio in the ellipticity indicates that peptide B forms twisted antiparallel beta-sheet conformation, whereas peptide A takes a parallel beta-sheet conformation. The results obtained in this work indicates the role of polar derivatization on the conformational preference of peptides having similar sequence.     

Expression of an antibody Fv fragment in myeloma cells

The antigen binding site on antibodies is fashioned by loops at the tips of the beta-sheet framework of both heavy and light chain variable domains. A heterodimer of both variable domains (Fv fragment), incorporating loops from an anti-lysozyme antibody, was expressed and secreted from myeloma cells in good yield (8 mg/l in supernatant from roller bottles), and shown to bind lysozyme. The two subunits were found to be in dynamic equilibrium but are overwhelmingly associated at neutral pH. The small size of Fv fragments (25 x 10(3) Mr) make them attractive for structural studies, in vivo imaging, and therapy.     

Comparison of the structural stability of two homologous toxins isolated from the Taiwan cobra (Naja naja atra) venom

Cardiotoxin analogue III (CTX III) and cobrotoxin (CBTX) isolated from the Taiwan cobra venom (Naja naja atra) are structurally homologous, small molecular weight, all-beta-sheet proteins, cross-linked by four disulfide bonds at identical positions. The conformational stabilities of these toxins are compared based on temperature-dependent chemical shifts and amide proton exchange kinetics using two-dimensional NMR spectroscopy. The structure of CTX III is found to be significantly more stable than that of CBTX. In both the toxins, beta-strand III appears to constitute the stability core. In CTX III, the stability of the triple-stranded beta-sheet domain is observed to be markedly higher than the double-stranded beta-sheet segment. In contrast, in CBTX, both structural domains (double- and triple-stranded beta-sheet domains) appear to contribute equally to the stability of the protein. Estimation of the free energy of exchange (Delta G(ex)) of residues in CBTX and CTX III reveals that the enhanced stability of the structure of CTX III stems from the strong interactions among the beta-strands constituting the triple-stranded beta-sheet domain and also the molecular forces bridging the residues at the N- and C-terminal ends of the molecule.     

Plant biodiversity inventory and conservation of two tropical dry evergreen forests on the Coromandel coast, south India

Species diversity, population structure, abundance and dispersion patterns of all woody plants 10cm gbh were inventoried in two 1-ha plots of tropical dry evergreen (sacred grove or temple) forests at Kuzhanthaikuppam (KK) and Thirumanikkuzhi (TM) on the Coromandel coast of south India. Site KK is a stunted forest (average tree height ca 6 m) and TM a tall forest (average tree height ca 10 m). A total of 54 species (in 47 genera and 31 families) were recorded. Species richness and stand density were 42 and 38 species and 1367 and 974 individuals ha–1 respectively for the sites KK and TM. About 50% of the total species were common to both the sites. Site TM is twofold more voluminous (basal area 29.48 m2 ha–1) than KK (basal area 15.44 m2 ha–1). Nearly one third of the individuals are multi-stemmed in the low-statured site KK whereas one fourth of the tree density is multi-stemmed in TM. Species abundance pattern varied between the two sites. The abundance of three species in KK and two species in TM is pronounced. Memecylon umbellatum, the most abundant species contributing to one third of total stand density in KK, is least represented in TM. Species richness, density and diversity indices decreased with increasing girth threshold. Most species exhibited clumped dispersion of individuals both at 0.25 and 1-ha scales. Population structure for girth frequency is an expanding one for both the sites, except for basal area distribution in KK. Variations in plant diversity and abundance are related to site attributes and human impacts. In the light of habitat uniqueness, species richness and sacred grove status, the need for conservation is emphasized.     

Strand-loop-strand motifs: prediction of hairpins and diverging turns in proteins

Beta-sheet proteins have been particularly challenging for de novo structure prediction methods, which tend to pair adjacent beta-strands into beta-hairpins and produce overly local topologies. To remedy this problem and facilitate de novo prediction of beta-sheet protein structures, we have developed a neural network that classifies strand-loop-strand motifs by local hairpins and nonlocal diverging turns by using the amino acid sequence as input. The neural network is trained with a representative subset of the Protein Data Bank and achieves a prediction accuracy of 75.9 +/- 4.4% compared to a baseline prediction rate of 59.1%. Hairpins are predicted with an accuracy of 77.3 +/- 6.1%, diverging turns with an accuracy of 73.9 +/- 6.0%. Incorporation of the beta-hairpin/diverging turn classification into the ROSETTA de novo structure prediction method led to higher contact order models and somewhat improved tertiary structure predictions for a test set of 11 all-beta-proteins and 3 alphabeta-proteins. The beta-hairpin/diverging turn classification from amino acid sequences is available online for academic use (Meiler and Kuhn, 2003; www.jens-meiler.de/turnpred.html).     

Biodegradable Injectable In Situ Implants and Microparticles for Sustained Release of Montelukast: In Vitro Release,Pharmacokinetics, and Stability

The objective of this study was to investigate the sustained release of a hydrophilic drug, montelukast (MK), from two biodegradable polymeric drug delivery systems, in situ implant (ISI) and in situ microparticles (ISM). N-Methyl pyrrolidone (NMP), dimethyl sulfoxide (DMSO), triacetin, and ethyl acetate were selected as solvents. The release of 10% (w/v) MK from both systems containing poly-lactic-co-glycolic acid (PLGA) as the biodegradable polymer was compared. Upon contact with the aqueous medium, the PLGA in ISI and ISM systems solidified resulting in implants and microparticles, respectively. The in vitro drug release from the ISI system showed marked difference from miscible solvents (NMP and DMSO) than the partially miscible ones (triacetin and ethyl acetate), and the drug release decreased with increased PLGA concentration. In the ISM system, the initial in vitro drug release decreased with decreased ratio of polymer phase to external oil phase. In vivo studies in rats showed that ISM had slower drug release than the drug release from ISI. Also, the ISM system when compared to ISI system had significantly reduced initial burst effect. In vitro as well as the in vivo studies for both ISI and ISM systems showed sustained release of MK. The ISM system is suitable for sustained release of MK over 4-week period with a lower initial burst compared to the ISI system. Stability studies of the ISI and ISM formulations showed that MK is stable in the formulations stored at 4°C for more than 2 years.     

Artificial model of photosynthetic oxygen evolving complex: catalytic O2 production from water by di-mu-oxo manganese dimers supported by clay compounds

Adsorption of [(OH(2))(terpy)Mn(mu-O)(2)Mn(terpy)(OH(2))](3+) (terpy=2,2':6',2"-terpyridine) (1) onto montmorillonite K10 (MK10) yielded catalytic dioxygen (O(2)) evolution from water using a Ce(IV) oxidant. The Mn K-edge X-ray absorption near edge structure (XANES) of the 1/MK10 hybrid suggested that the oxidation state of the di-mu-oxo Mn(2) core could be Mn(III)-Mn(IV). However the pre-edge peak in the XANES spectrum of 1 adsorbed on MK10 is different from the neat 1 powder. The kinetic analysis of O(2) evolution showed that the catalysis requires cooperation of two equivalents of 1 adsorbed on MK10. The reaction of the [(bpy)(2)Mn(mu-O)(2)Mn(bpy)(2)](3+) (bpy=2,2'-bipyridine) (2)/MK10 hybrid with a Ce(IV) oxidant evolved O(2). However, the turnover number value was less than unity for 2/MK10, showing that 2 adsorbed on MK10 does not work as a catalyst. The terminal water ligands could be an important for the catalysis by adsorbed 1. The mechanism of O(2) production by photosynthetic oxygen evolving complex is discussed based on catalytic O(2) evolution by 1 adsorbed on MK10.     

Hydrogen bonding and equilibrium protium-deuterium fractionation factors in the immunoglobulin G binding domain of protein G

Protium-deuterium fractionation factors (phi) were determined for more than 85% of the backbone amide protons in the IgG binding domains of protein G, GB1 and GB2, from NMR spectra recorded over a range of H2O/D2O solvent ratios. Previous studies suggest a correlation between phi and hydrogen bond strength; amide and hydroxyl groups in strong hydrogen bonds accumulate protium (phi < 1), while weak hydrogen bonds accumulate deuterium (phi > 1). Our results show that the alpha-helical residues have slightly lower phi values (1.03 +/- 0.05) than beta-sheet residues (1.12 +/- 0.07), on average. The lowest phi value obtained (0.65) does not involve a backbone amide but rather is for the interaction between two side chains, Y45 and D47. Fractionation factors for solvent-exposed residues are between the alpha-helix and beta-sheet values, on average, and are close to those for random coil peptides. Further, the difference in phiav between alpha-helix and solvent-exposed residues is small, suggesting that differences in hydrogen bond strength for intrachain hydrogen bonds and amide...water hydrogen bonds are also small. Overall, the enrichment for deuterium suggests that most backbone...backbone hydrogen bonds are weak.     

The predicted secondary structure of enolase.

The results of several secondary-structure prediction programs were combined to produce an estimate of the regions of alpha-helix, beta-sheet and reverse turn for both chicken skeletal-muscle and yeast enolase sequences. The predicted secondary-structure content of the chicken enzyme is 27% alpha-helix and less than 10% beta-sheet, whereas in the yeast enolase a similar helix content but virtually no sheet are predicted. These results are in fair agreement with published experimental estimates of the amount of secondary structure in the yeast enzyme. The enzyme appears to be formed from three domains.     

Water-soluble beta-sheet models which self-assemble into fibrillar structures.

Self-assembly of beta-sheet domains resulting in the formation of pathogenic, fibrillar protein aggregates (amyloids) is a characteristic feature of various medical disorders. These include neurodegenerative diseases, such as Alzheimer's, Huntington's, and Creutzfeldt-Jacob's. A significant problem in studying such aggregation processes is the poor solubility of these beta-sheet complexes. The present work describes water-soluble de novo beta-sheet peptides which self-assemble into fibrillar structures. The model peptides enable studies of the relationship between beta-sheet stability and association behavior. The peptides [DPKGDPKG-(VT)n-GKGDPKPD-NH2, n = 3-8] are composed of a central beta-sheet-forming domain (VT-sequence), and N- and C-terminal nonstructured octapeptide sequences which promote water solubility. Conformational analyses by circular dichroism and Fourier transform infrared spectroscopy indicate the influence of peptide length, D-amino acid substitution, and concentration on the ability of the peptides to form stable beta-sheet structures. The association behavior investigated by analytical ultracentrifugation and dynamic light scattering was found to correlate strongly with the stability of a beta-sheet conformation. Model peptides with n >/= 6 form stable, water-soluble beta-sheet complexes with molecular masses of more than 2000 kDa, which are organized in fibrillar structures. The fibrils examined by Congo Red staining and electron microscopy show some similarities with naturally occurring amyloid fibrils.     

Stability of the beta-sheet of the WW domain: A molecular dynamics simulation study.

The WW domain consists of approximately 40 residues, has no disulfide bridges, and forms a three-stranded antiparallel beta-sheet that is monomeric in solution. It thus provides a model system for studying beta-sheet stability in native proteins. We performed molecular dynamics simulations of two WW domains, YAP65 and FBP28, with very different stability characteristics, in order to explore the initial unfolding of the beta-sheet. The less stable YAP domain is much more sensitive to simulation conditions than the FBP domain. Under standard simulation conditions in water (with or without charge-balancing counterions) at 300 K, the beta-sheet of the YAP WW domain disintegrated at early stages of the simulations. Disintegration commenced with the breakage of a hydrogen bond between the second and third strands of the beta-sheet due to an anticorrelated transition of the Tyr-28 psi and Phe-29 phi angles. Electrostatic interactions play a role in this event, and the YAP WW domain structure is more stable when simulated with a complete explicit model of the surrounding ionic strength. Other factors affecting stability of the beta-sheet are side-chain packing, the conformational entropy of the flexible chain termini, and the binding of cognate peptide.     

Two-dimensional structure of beta-amyloid(10-35) fibrils

Beta-amyloid (Abeta) peptides are the main protein component of the pathognomonic plaques found in the brains of patients with Alzheimer's disease. These heterogeneous peptides adopt a highly organized fibril structure both in vivo and in vitro. Here we use solid-state NMR on stable, homogeneous fibrils of Abeta(10-35). Specific interpeptide distance constraints are determined with dipolar recoupling NMR on fibrils prepared from a series of singly labeled peptides containing (13)C-carbonyl-enriched amino acids, and skipping no more that three residues in the sequence. From these studies, we demonstrate that the peptide adopts the structure of an extended parallel beta-sheet in-register at pH 7.4. Analysis of DRAWS data indicates interstrand distances of 5.3 +/- 0.3 A (mean +/- standard deviation) throughout the entire length of the peptide, which is compatible only with a parallel beta-strand in-register. Intrastrand NMR constraints, obtained from peptides containing labels at two adjacent amino acids, confirm the secondary structural findings obtained using DRAWS. Using peptides with (13)C incorporated at the carbonyl position of adjacent amino acids, structural transitions from alpha-helix to beta-sheet were observed at residues 19 and 20, but using similar techniques, no evidence for a turn could be found in the putative turn region comprising residues 25-29. Implications of this extended parallel organization for Abeta(10-35) for overall fibril formation, stability, and morphology based upon specific amino acid contacts are discussed.     

Structural comparison of the unstable drkN SH3 domain and a stable mutant

The N-terminal SH3 domain of the Drosophila adapter protein Drk (drkN SH3 domain) is marginally stable (DeltaG(U) = 1 kcal/mol) and exists in equilibrium between folded and highly populated unfolded states. The single substitution T22G, however, completely stabilizes the protein (DeltaG(U) = 4.0 kcal/mol). To probe the causes of instability of the wild-type (WT) protein and the dramatic stabilization of the mutant, we determined and compared nuclear magnetic resonance structures of the folded WT and mutant drkN SH3 domains. Residual dipolar coupling (RDC) and carbonyl chemical-shift anisotropy (C'-CSA) restraints measured for the WT and T22G domains were used for calculating the structures. The structures for the WT and mutant are highly similar. Thr22 of the WT and Gly22 of the mutant are at the i + 2 position of the diverging, type-II beta-turn. Interestingly, not only Gly22 but also Thr22 successfully adopt an alpha(L) conformation, required at this position of the turn, despite the fact that positive phi values are energetically unfavorable and normally disallowed for threonine residues. Forcing the Thr22 residue into this unnatural conformation increases the free energy of the folded state of the WT domain relative to its T22G mutant. Evidence for residual helix formation in the diverging turn region has been previously reported for the unfolded state of the WT drkN SH3 domain, and this, in addition to other residual structure, has been proposed to play a role in decreasing the free energy of the unfolded state of the protein. Together these data provide evidence that both increasing the free energy of the folded state and decreasing the free energy of the unfolded state of the protein contribute to instability of the WT drkN SH3 domain.     

Design and NMR conformational study of a beta-sheet peptide based on Betanova and WW domains

A good approach to test our current knowledge on formation of protein beta-sheets is de novo protein design. To obtain a three-stranded beta-sheet mini-protein, we have built a series of chimeric peptides by taking as a template a previously designed beta-sheet peptide, Betanova-LLM, and incorporating N- and/or C-terminal extensions taken from WW domains, the smallest natural beta-sheet domain that is stable in absence of disulfide bridges. Some Betanova-LLM strand residues were also substituted by those of a prototype WW domain. The designed peptides were cloned and expressed in Escherichia coli. The ability of the purified peptides to adopt beta-sheet structures was examined by circular dichroism (CD). Then, the peptide showing the highest beta-sheet population according to the CD spectra, named 3SBWW-2, was further investigated by 1H and 13C NMR. Based on NOE and chemical shift data, peptide 3SBWW-2 adopts a well defined three-stranded antiparallel beta-sheet structure with a disordered C-terminal tail. To discern between the contributions to beta-sheet stability of strand residues and the C-terminal extension, the structural behavior of a control peptide with the same strand residues as 3SBWW-2 but lacking the C-terminal extension, named Betanova-LYYL, was also investigated. beta-Sheet stability in these two peptides, in the parent Betanova-LLM and in WW-P, a prototype WW domain, decreased in the order WW-P > 3SBWW-2 > Betanova-LYYL > Betanova-LLM. Conclusions about the contributions to beta-sheet stability were drawn by comparing structural properties of these four peptides.     

Structure of the C-terminal domain from Trypanosoma brucei variant surface glycoprotein MITat1.2

The variant surface glycoprotein (VSG) of African trypanosomes has a structural role in protecting other cell surface proteins from effector molecules of the mammalian immune system and also undergoes antigenic variation necessary for a persistent infection in a host. Here we have reported the solution structure of a VSG type 2 C-terminal domain from MITat1.2, completing the first structure of both domains of a VSG. The isolated C-terminal domain is a monomer in solution and forms a novel fold, which commences with a short alpha-helix followed by a single turn of 3(10)-helix and connected by a short loop to a small anti-parallel beta-sheet and then a longer alpha-helix at the C terminus. This compact domain is flanked by two unstructured regions. The structured part of the domain contains 42 residues, and the core comprises 2 disulfide bonds and 2 hydrophobic residues. These cysteines and hydrophobic residues are conserved in other VSGs, and we have modeled the structures of two further VSG C-terminal domains using the structure of MITat1.2. The models suggest that the overall structure of the core is conserved in the different VSGs but that the C-terminal alpha-helix is of variable length and depends on the presence of charged residues. The results provided evidence for a conserved tertiary structure for all the type 2 VSG C-terminal domains, indicated that VSG dimers form through interactions between N-terminal domains, and showed that the selection pressure for sequence variation within a conserved tertiary structure acts on the whole of the VSG molecule.     

Formation of the single-layer beta-sheet of Borrelia burgdorferi OspA in the absence of the C-terminal capping globular domain

Borrelia outer surface protein A (OspA) contains a unique single-layer beta-sheet that connects N and C-terminal globular domains. This single-layer beta-sheet segment (beta-strands 8-10) is highly stable in solution, although it is exposed to the solvent on both faces of the sheet and thus it does not contain a hydrophobic core. Here, we tested whether interactions with the C-terminal domain are essential for the formation of the single-layer beta-sheet. We characterized the solution structure, dynamics and stability of an OspA fragment corresponding to beta-strands 1-12 (termed OspA[27-163]), which lacks a majority of the C-terminal globular domain. Analyses of NMR chemical shifts and backbone nuclear Overhauser effect (NOE) connectivities showed that OspA[27-163] is folded except the 12th and final beta-strand. (1)H-(15)N heteronuclear NOE measurements and amide H-(2)H exchange revealed that the single-layer beta-sheet in this fragment is more flexible than the corresponding region in full-length OspA. Thermal-denaturation experiments using differential scanning calorimetry and NMR spectroscopy revealed that the N-terminal globular domain in the fragment has a conformational stability similar to that of the same region in the full-length protein, and that the single-layer beta-sheet region also has a modest thermal stability. These results demonstrate that the unique single-layer beta-sheet retains its conformation in the absence of its interactions with the C-terminal domain. This fragment is significantly smaller than the full-length OspA, and thus it is expected to facilitate studies of the folding mechanism of this unusual beta-sheet structure.