A CD strategy for the study of polypeptide folding/unfolding. A synthetic foot-and-mouth disease virus immunogenic peptide.

The circular dichroism spectrum of the 20-residue immunogenic peptide from the foot-and-mouth disease virus (VP1; 141-160 of serotype A, subtype 12) was solvent- and temperature-dependent. Careful solvent titration revealed two isodichroic points and plateaux consistent with stepwise unfolding of specific stable conformations. Variable temperature studies in cryogenic solvents and urea perturbation were consistent with the existence of three conformational moieties, the left-handed extended helix, the alpha-helix, and the 3(10) helix. The number of residues in each helix was confirmed by CD spectral simulations. The strategy described here can be used to determine the components of a conformational equilibrium and their statistical weights, to study peptide folding and unfolding and to determine the bioactive conformation(s) of linear peptides. The conclusions were supported by 2D-NMR studies. A new mechanism for the stabilization of left-handed extended helices and destabilization of alpha-helices by urea is proposed. The structure of the peptide as resolved by CD spectroscopy is of particular significance since the conformation of this antigenic sequence in situ has so far not been solved by X-ray crystallography.     

Facile transition between 3(10)- and alpha-helix: structures of 8-, 9-, and 10-residue peptides containing the -(Leu-Aib-Ala)2-Phe-Aib- fragment.

A structural transition from a 3(10)-helix to an alpha-helix has been characterized at high resolution for an octapeptide segment located in 3 different sequences. Three synthetic peptides, decapeptide (A) Boc-Aib-Trp-(Leu-Aib-Ala)2-Phe-Aib-OMe, nonapeptide (B) Boc-Trp-(Leu-Aib-Ala)2-Phe-Aib-OMe, and octapeptide (C) Boc-(Leu-Aib-Ala)2-Phe-Aib-OMe, are completely helical in their respective crystals. At 0.9 A resolution, R factors for A, B, and C are 8.3%, 5.4%, and 7.3%, respectively. The octapeptide and nonapeptide form ideal 3(10)-helices with average torsional angles phi(N-C alpha) and psi(C alpha-C') of -57 degrees, -26 degrees C and -60 degrees, -27 degrees for B. The 10-residue peptide (A) begins as a 3(10)-helix and abruptly changes to an alpha-helix at carbonyl O(3), which is the acceptor for both a 4-->1 hydrogen bond with N(6)H and a 5-->1 hydrogen with N(7)H, even though the last 8 residues have the same sequence in all 3 peptides. The average phi, psi angles in the decapeptide are -58 degrees, -28 degrees for residues 1-3 and -63 degrees, -41 degrees for residues 4-10. The packing of helices in the crystals does not provide any obvious reason for the transition in helix type. Fourier transform infrared studies in the solid state also provide evidence for a 3(10)- to alpha-helix transition with the amide I band appearing at 1,656-1,657 cm-1 in the 9- and 10-residue peptides, whereas in shorter sequences the band is observed at 1,667 cm-1.     

Synthesis and spectroscopy of membrane receptor proteins. The gamma subunit of the IgE receptor.

The high-affinity receptor for IgE is a tetrameric complex of subunits of the type alpha beta gamma 2. We report here conformational studies of the intact gamma subunit in trifluoroethanol and water/liposomes by circular dichroism and Fourier-transform infrared (FTIR) spectroscopy. In trifluoroethanol, the FTIR amide I' frequencies were consistent with two predominant conformational components, the beta-turn and alpha-helix, whilst in liposomes consisting of D2O and dimyristoylglycerophosphocholine (Myr2GroPCho), three components were observed. The third component present may contain some left-handed extended helix. Spectral simulation was carried out to demonstrate that the CD spectra were consistent with the component conformations identified from FTIR spectroscopy. The stimulated CD spectra were in excellent agreement with the experimental spectra. The intact gamma subunit conformation in trifluoroethanol was shown to possess 72% alpha-helical and 28% beta-turn conformations. In water/Myr2GroPCho liposomes the percentage of each conformational component present is 37%, 38% and 25% for the alpha-helix, beta-turn and extended structures, respectively. Assuming that the transmembrane fragment was alpha-helical, an excellent correlation was found between this derived alpha-helical content in water/liposomes (37%) and from hydrophobicity plots where the percentage of amino acids in the transmembrane domain is predicted by others to be 34%. It is suggested that the beta-turn detected by CD and FTIR was attributable to a 3(10) helix rather than a type I or type III reverse turn.     

Conformational studies of Aib-rich peptides containing lactam-bridged side chains: evidence of 3(10)-helix formation

Aib-rich side-chain lactam-bridged oligomers Ac-(Glu-Aib-Aib-Lys)n-Ala-OH with n = 1,2,3 were designed and synthesized as putative models of the 3(10)-helix. The lactam bridge between the side chains of L-Glu and L-Lys in (i)--(i + 3) positions was introduced in order to enhance the structural preference toward the right-handed 3(10)-helix. The conformational properties of the three peptides were studied in trifluoroethanol (TFE) solution by CD, NMR, and computer simulations. The structural information was derived mainly from the analysis of nuclear Overhauser effect spectroscopy spectra. The presence of alpha H(i)-HN(i + 2) and of alpha H(i)-HN(i + 3) connectivities and the absence of alpha H(i)-HN(i + 4) connectivities indicate that these peptides fold into a 3(10)-helix rather than into an alpha-helix. Based on these conformational features, stereospecific assignment of the Aib methyl groups was possible. The results of such experiments and of the subsequent distance geometry and restrained molecular dynamics simulations reveal a marked preference of these peptides for 3(10)-helix. The CD spectra of these peptides indicate that the helix content increases upon chain elongation. The CD spectrum of the trimer is characterized by a negative band at 200 nm and by a weak positive band around 220 nm. The CD spectrum in TFE is different from that observed in aqueous solution in the presence of SDS micelles, reported in our previous work, and from those reported by a different research group for 3(10)-helical peptides. A possible reason for these differences could rest in the presence of different equilibria of the conformer populations of the various peptides in different solvent systems.     

Structure of the propeptide of prothrombin containing the gamma-carboxylation recognition site determined by two-dimensional NMR spectroscopy

The propeptides of the vitamin K dependent blood clotting and regulatory proteins contain a gamma-carboxylation recognition site that directs precursor forms of these proteins for posttranslational gamma-carboxylation. Peptides corresponding to the propeptide of prothrombin were synthesized and examined by circular dichroism (CD) and nuclear magnetic resonance spectroscopy (NMR). CD spectra indicate that these peptides have little or no secondary structure in aqueous solutions but that the addition of trifluoroethanol induces or stabilizes a structure containing alpha-helical character. The maximum helical content occurs at 35-40% trifluoroethanol. This trifluoroethanol-stabilized structure was solved by two-dimensional NMR spectroscopy. The NMR results demonstrate that residues -13 to -3 form an amphipathic alpha-helix. NMR spectra indicate that a similar structure is present at 5 degrees C, in the absence of trifluoroethanol. Of the residues previously implicated in defining the gamma-carboxylation recognition site, four residues (-18, -17, -16, and -15) are adjacent to the helical region and one residue (-10) is located within the helix. The potential role of the amphipathic alpha-helix in the gamma-carboxylation recognition site is discussed.     

Low-ultraviolet circular dichroism spectroscopy of oligopeptides 1-95 and 96-168 derived from myelin basic protein of rabbit

Myelin basic protein (MBP) is a major protein constituent of the myelin sheath of the central nervous system, where it is believed to have functional alpha-helical segments. One element of the function of the protein might be "conformational adaptability" of specific regions of its amino acid sequence, since the purified protein appears to be largely devoid of ordered structure. To pursue this question, low-ultraviolet circular dichroism (CD) spectroscopy was conducted on the sequential thrombic peptides 1-95 and 96-168 of the protein in the presence of 0-92% trifluoroethanol (TFE), a solvent known to promote stable secondary structures in polypeptides. The series of CD spectra of the oligopeptides were subjected to a computerized best-fit analysis of four peptide conformations, the alpha-helix, beta-structure, beta-turn, and nonordered form. Agreement between experimental and best-fit composite spectra was achieved when standard CD curves of peptide conformations were derived from known theoretical spectra and experimental spectra of polypeptides. In dilute buffer alone, oligopeptides 1-95 and 96-168 evidence no alpha-helix but significant beta-structure (18% and 23%, respectively), as well as a predominant, extended nonordered conformation. However, the two parts of the protein differed in conformational adaptability. From 0% to 30% TFE, 96-168 exhibited concomitant transitions to 10% helix and 32% beta-structure from the nonordered form. In contrast, in 10-30% TFE, 1-95 underwent a transition to approximately 21% helix with partial loss of beta-structure as well as nonordered form; higher concentrations of TFE (40-75%) promoted additional transitions to both helix and beta-structure (totaling 33% and 25%, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization at atomic resolution of peptide helical structures.

A survey of literature for the various types of helices experimentally observed in high-resolution single crystal x-ray diffraction analyses of peptides has allowed to determine accurate conformational and helical parameters for the various secondary structures such as the alpha-helix, the 3(10)-helix, the fully extended conformation (2(5)-helix) and the beta-bend ribbon spiral. For each of these structures the characteristic phi, psi conformational parameters, n, the number of residues per turn, h, the height per residues and p, the pitch of the helix are described.     

Helix aggregation in peptide crystals: occurrence of either all parallel or antiparallel packing motifs for alpha-helices in polymorphs of Boc-Aib-Ala-Leu-Ala-Leu-Aib-Leu-Ala-Leu-Aib-OMe

Three crystalline polymorphs of the helical decapeptide, Boc-Aib-Ala-Leu-Ala-Leu-Aib-Leu-Ala-Leu-Aib-OMe, have been obtained. Antiparallel helix aggregation is observed in crystals grown from methanol (A), while completely parallel packing is observed in crystals from isopropanol (B) or an ethylene glycol-ethanol mixture (C). Crystals B and C are very similar in molecular conformation and packing. The packing motifs in crystals A and B consist of rows of parallel molecules, with an almost identical arrangement in both crystals. In crystal A, adjacent rows assemble with the helix axes pointed in opposite directions, whereas in crystal B all rows assemble with helix axes pointed in the same direction. Electrostatic interactions between helix dipoles do not appear to be a major determinant of packing modes. The structures also do not provide a ready rationalization of packing preferences in terms of side-chain interactions or solvation. The alpha-helix of the peptide in crystal A has seven 5----1 hydrogen bonds; the helix in crystal B is a mixed 3(10)/alpha-helix. The crystal parameters are as follows. Crystal A: C51H92N10O13.CH3OH, space group P2(1) with a = 10.498 (1) A, b = 18.189 (3) A, c = 16.475 (3) A, beta = 99.28 (1) degree, Z = 2, R = 9.6% for 1860 data. Crystal B: C51H92N10O13.C3H7OH, space group P2(1) with a = 10.534 (1) A, b = 28.571 (4) A, c = 11.055 (2) A, beta = 95.74 (1) degree, Z = 2, R = 6.5% for 3251 data. Crystal C: C51H92N10O13.C2H5OH, space group P2(1), with a = 10.450 (1) A, b = 28.442 (5) A, c = 11.020 (2) A, beta = 95.44(1) degree, Z = 2, R = 14.8% (isotropic) for 1948 data.     

The T lymphocyte response to cytochrome c. V. Determination of the minimal peptide size required for stimulation of T cell clones and assessment of the contribution of each residue beyond this size to antigenic potency

The B10.A T cell proliferative response to pigeon cytochrome c is mainly directed against a single antigenic determinant located at the carboxy-terminal end of the molecule. In the present experiments, we used synthetic peptide analogs of the carboxy-terminal sequence of moth cytochrome c to explore the structural requirements for antigenic potency. The minimum-sized peptide capable of stimulating a full response varied with the T cell clone, but within the limits of the biological systems studied, was shown to be moth fragment 97-103. Addition of more amino acids at the amino terminal end increased the antigenic potency in uneven increments, with a large contribution being made at residue 95. Analysis of amino acid substitutions at this position provided no evidence that it contained a residue that directly contacted the T cell receptor. Instead, good agreement with an analysis that made use of helix-coil transition theory suggested that this residue, as well as others, increased antigenic potency by contributing to the stabilization of the secondary structure of the molecule in an alpha-helical configuration. The maximum effect of chain length on antigenic potency appeared to stop at residue 93, in agreement with the theoretical analysis. However, addition of several more amino-terminal residues to residue 93 showed one additional significant increment of increased potency. This was almost entirely accounted for by a single lysine located four amino acids beyond the glutamic acid at residue 93 (approximately one turn of an alpha-helix away). To experimentally test whether alpha-helix-forming tendencies could account for the increased potency of the larger analogs, the degree of helix formation in trifluoroethanol was assessed by circular dichroism measurements. A good correlation was found between antigenic potency and percentage of alpha-helix for peptides of increasing chain length from moth 95-103 up to moth 86-90; 94-103. These results suggest that secondary structure may play an important role in determining the potency of antigenic determinants involved in the activation of T lymphocytes.     

Hyperphosphorylation of tau induces local polyproline II helix

Alzheimer's disease is characterized by two protein precipitates, extracellular amyloid plaques and intracellular neurofibrillary tangles (NFTs). The primary constituent of NFTs is a hyperphosphorylated form of the microtubule-binding protein tau. Hyperphosphorylation of tau on over 30 residues, primarily within proline-rich sequences, is associated with conformational changes whose nature is poorly defined. Peptides derived from the proline-rich region of tau (residues 174-242) were synthesized, and the conformations were analyzed for the nonphosphorylated and phosphorylated peptides. CD and NMR data indicate that phosphorylation of serine and threonine residues in proline-rich sequences induces a conformational change to a type II polyproline helix. The largest phosphorylation-dependent conformational changes observed by CD were for tau peptides incorporating residues 174-183 or residues 229-238. Phosphoserine and phosphothreonine residues exhibited ordered values of (3)J(alphaN) (3.1-6.2 Hz; mean = 4.7 Hz) compared to nonphosphorylated serine and threonine. Phosphorylation of a tau peptide consisting of tau residues 196-209 resulted in the disruption of a nascent alpha-helix. These results suggest that global reorganization of tau may occur upon hyperphosphorylation of proline-rich sequences in tau.     

Folding of a peptide corresponding to the alpha-helix in bovine pancreatic trypsin inhibitor

A short peptide corresponding to the alpha-helical region of BPTI shows partial folding in aqueous solution (pH 7) as judged by circular dichroism (CD). Folding is temperature and denaturant sensitive, and the peptide is monomeric. The difference CD spectrum, obtained from spectra at two temperatures, indicates that the peptide folds as an alpha-helix. Difference CD spectroscopy provides a sensitive assay for helix formation in peptides exhibiting small amounts of structure. Helix stability in this peptide shows a marked pH dependence which is consistent with stabilizing charged side-chain interactions with the helix dipole and/or salt bridge formation.     

Synthesis and study of peptides with semirigid i and i + 7 side-chain bridges designed for alpha-helix stabilization

A search for conformational constraints on the peptide alpha-helical conformation indicated that para-substituted amino acid derivatives of a benzene ring might be suitable for linking pairs of side chains that are separated by two turns of the helix. A 14-residue synthetic, amphiphilic alpha-helical peptide model system has been used to study the helix stabilizing effects of a series of four such bridges having constitutionally isomeric structures. These bridges were used to link positions 3 and 10 of the model peptides. The peptides were synthesized in good yield by standard solid-phase methods, including cyclization on the solid support. They were then studied for their solution conformations and melting behavior by circular dichroism (CD) spectropolarimetry, and for their elution behavior on reversed-phase HPLC columns. In aqueous solution and in 50% (v/v) trifluoroethanol, the most effective bridge for helix stabilization consisted of a 4-(aminomethyl)phenylacetic acid residue (AMPA) linked by amide bonds to the side chain functional groups of a (S)-2,3-diaminopropionic acid residue (Dap) in position 3 of the model peptide and an aspartic acid residue in position 10. This Dap3(AMPA), Asp10 bridge was about as effective as two Lys(i), Asp(i+4) lactam bridges incorporated linking residues 3 and 7, and 10 and 14, in the same model peptide sequence. This suggests that it is worth about 1 kcal/mol of helix stabilization energy.     

Three-dimensional structure of the two peptides that constitute the two-peptide bacteriocin plantaricin EF

The three-dimensional structures of the two peptides plantaricin E (plnE; 33 residues) and plantaricin F (plnF; 34 residues) constituting the two-peptide bacteriocin plantaricin EF (plnEF) have been determined by nuclear magnetic resonance (NMR) spectroscopy in the presence of DPC micelles. PlnE has an N-terminal alpha-helix (residues 10-21), and a C-terminal alpha-helix-like structure (residues 25-31). PlnF has a long central alpha-helix (residues 7-32) with a kink of 38+/-7 degrees at Pro20. There is some flexibility in the helix in the kink region. Both helices in plnE are amphiphilic, while the helix in plnF is polar in its N-terminal half and amphiphilic in its C-terminal half. The alpha-helical content obtained by NMR spectroscopy is in agreement with CD studies. PlnE has two GxxxG motifs which are putative helix-helix interaction motifs, one at residues 5 to 9 and one at residues 20 to 24, while plnF has one such motif at residues 30 to 34. The peptides are flexible in these GxxxG regions. It is suggested that the two peptides lie parallel in a staggered fashion relative to each other and interact through helix-helix interactions involving the GxxxG motifs.     

Critical Main-Chain Length for Conformational Conversion From 3(10)-Helix to α-Helix in Polypeptides

To assess the minimal peptide length required for the stabilization of the alpha-helix relative to the 3(10)-helix in Aib-rich peptides, we have solved the X-ray diffraction structures of the terminally blocked sequential hexa- and octapeptides with the general formula-(Aib-L-Ala)n-(n = 3 and 4, respectively). The hexapeptide molecules are completely 3(10)-helical with four 1----4 intramolecular N-H . . . O = C H-bonds. On the other hand, the octapeptide molecules are essentially alpha-helical with four 1----5 H-bonds; however, the helix is elongated at the N-terminus, with two 1----4 H-bonds, giving these molecules a mixed alpha/3(10)-helical character. In both compounds the right-handed screw sense of the helix is dictated by the presence of the Ala residues of L-configuration. This study represents the first experimental proof for a 3(10)----alpha-helix conversion in the crystal state induced by peptide backbone lengthening only.     

Conformational analysis of peptides corresponding to all the secondary structure elements of protein L B1 domain: secondary structure propensities are not conserved in proteins with the same fold.

The solution conformation of three peptides corresponding to the two beta-hairpins and the alpha-helix of the protein L B1 domain have been analyzed by circular dichroism (CD) and nuclear magnetic resonance spectroscopy (NMR). In aqueous solution, the three peptides show low populations of native and non-native locally folded structures, but no well-defined hairpin or helix structures are formed. In 30% aqueous trifluoroethanol (TFE), the peptide corresponding to the alpha-helix adopts a high populated helical conformation three residues longer than in the protein. The hairpin peptides aggregate in TFE, and no significant conformational change occurs in the NMR observable fraction of molecules. These results indicate that the helical peptide has a significant intrinsic tendency to adopt its native structure and that the hairpin sequences seem to be selected as non-helical. This suggests that these sequences favor the structure finally attained in the protein, but the contribution of the local interactions alone is not enough to drive the formation of a detectable population of native secondary structures. This pattern of secondary structure tendencies is different to those observed in two structurally related proteins: ubiquitin and the protein G B1 domain. The only common feature is a certain propensity of the helical segments to form the native structure. These results indicate that for a protein to fold, there is no need for large native-like secondary structure propensities, although a minimum tendency to avoid non-native structures and to favor native ones could be required.     

Fluorescence-coupled CD conformational monitoring of filament formation of tau microtubule-binding repeat domain

To clarify the contribution of the three- or four-repeated peptide moiety in tau microtubule-binding domain (MBD) to paired helical filament (PHF) formation, conformational transition accompanied by heparin-induced filament formation was investigated stepwise for four repeat peptides (R1-R4), one three-repeated R1-R3-R4 peptide (3RMBD), and one four-repeated R1-R2-R3-R4 peptide (4RMBD) using a combination of thioflavin S fluorescence and circular dichroism (CD) measurements in a neutral buffer (pH 7.6). The comparison of the fluorescence profile of each repeat peptide with those of 3RMBD and 4RMBD showed the synergistic contribution of R1-R4 to PHF formation of MBD. The CD spectrum measured as a function of filament formation time indicates that: (i) two conformational transitions occur for the filament formations of R3 (from the random structure to the beta-sheet structure) and 3RMBD (from the random structure to the alpha-helix structure), (ii) the filament formations of R2 and 4RMBD proceed via the synchronized conformational transitions of the alpha-helix and random structures, and (iii) the filament formation of 4RMBD is dependent on the aggregation behavior of R2. These data are useful for elucidating the MBD conformational transition in tau PHF formation.     

Stable proline box motif at the N-terminal end of alpha-helices.

We describe a novel N-terminal alpha-helix local motif that involves three hydrophobic residues and a Pro residue (Pro-box motif). Database analysis shows that when Pro is the N-cap of an alpha-helix the distribution of amino acids in adjacent positions changes dramatically with respect to the average distribution in an alpha-helix, but not when Pro is at position N1. N-cap Pro residues are usually associated to Ile and Leu, at position N', Val at position N3 and a hydrophobic residue (h) at position N4. The side chain of the N-cap Pro packs against Val, while the hydrophobic residues at positions N' and N4 make favorable interactions. To analyze the role of this putative motif (sequence fingerprint hPXXhh), we have synthesized a series of peptides and analyzed them by circular dichroism (CD) and NMR. We find that this motif is formed in peptides, and that the accompanying hydrophobic interactions contribute up to 1.2 kcal/mol to helix stability. The fact that some of the residues in this fingerprint are not good N-cap and helix formers results in a small overall stabilization of the alpha-helix with respect to other peptides having Gly as the N-cap and Ala at N3 and N4. This suggests that the Pro-box motif will not specially contribute to protein stability but to the specificity of its fold. In fact, 80% of the sequences that contain the fingerprint sequence in the protein database are adopting the described structural motif, and in none of them is the helix extended to place Pro at the more favorable N1 position.     

Conformational analysis of a set of peptides corresponding to the entire primary sequence of the N-terminal domain of the ribosomal protein L9: evidence for stable native-like secondary structure in the unfolded state

There is considerable interest in the structure of the denatured state and in the role local interactions play in protein stability and protein folding. Studies of peptide fragments provide one method to assess local conformational preferences which may be present in the denatured state under native-like conditions. A set of peptides corresponding to the individual elements of secondary structure derived from the N-terminal domain of the ribosomal protein L9 have been synthesized. This small 56 residue protein adopts a mixed alpha-beta topology and has been shown to fold rapidly in an apparent two-state fashion. The conformational preferences of each peptide have been analyzed by proton nuclear magnetic resonance spectroscopy and circular dichroism spectroscopy. Peptides corresponding to each of the three beta-stands and to the first alpha-helix are unstructured as judged by CD and NMR. In contrast, a peptide corresponding to the C-terminal helix is remarkably structured. This 17 residue peptide is 53 % helical at pH 5.4, 4 degrees C. Two-dimensional NMR studies demonstrate that the helical structure is distributed approximately uniformly throughout the peptide, although there is some evidence for fraying at the C terminus. Detailed analysis of the NMR spectra indicate that the helix is stabilized, in part, by a native N-capping interaction involving Thr40. A mutant peptide which lacks Thr40 is only 32 % helical. pH and ionic strength-dependent studies suggested that charge charge interactions make only a modest net contribution to the stability of the peptide. The protein contains a trans proline peptide bond located at the first position of the C-terminal helix. NMR analysis of the helical peptide and of a smaller peptide containing the proline residue indicates that only a small amount of cis proline isomer (8 %) is likely to be populated in the unfolded state.     

Conformational preferences of a short Aib/Ala‐based water‐soluble peptide as a function of temperature

The amino acid Aib predisposes a peptide to be helical with context‐dependent preference for either 3₁₀‐ or α‐ or a mixed helical conformation. Short peptides also show an inherent tendency to be unfolded. To characterize helical and unfolded states adopted by water‐soluble Aib‐containing peptides, the conformational preference of Ac‐Ala‐Aib‐Ala‐Lys‐Ala‐Aib‐Lys‐Ala‐Lys‐Ala‐Aib‐Tyr‐NH₂ was determined by CD, NMR and MD simulations as a function of temperature. Temperature‐dependent CD data indicated the contribution of two major components, each an admixture of helical and extended/polyproline II structures. Both right‐ and left‐handed helical conformations were detected from deconvolution of CD data and ¹³C NMR experiments. The presence of a helical backbone, more pronounced at the N‐terminal, and a temperature‐induced shift in α‐helix/3₁₀‐helix equilibrium, more pronounced at the C‐terminal, emerged from NMR data. Starting from polyproline II, the N‐terminal of the peptide folded into a helical backbone in MD simulations within 5 ns at 60°C. Longer simulations showed a mixed‐helical backbone to be stable over the entire peptide at 5°C while at 60°C the mixed‐helix was either stable at the N‐terminus or occurred in short stretches through out the peptide, along with a significant population of polyproline II. Our results point towards conformational heterogeneity of water‐soluble Aib‐based peptide helices and the associated subtleties. The problem of analyzing CD and NMR data of both left‐ and right‐handed helices are discussed, especially the validity of the ellipticity ratio [θ]₂₂₂/[θ]₂₀₇, as a reporter of α‐/3₁₀‐ population ratio, in right‐ and left‐handed helical mixtures. Proteins 2009. © 2008 Wiley‐Liss, Inc.     

Role of listeriolysin-O (LLO) in the T lymphocyte response to infection with Listeria monocytogenes. Identification of T cell epitopes of LLO.

Using a murine model, we investigated the role of the bacterial exotoxin listeriolysin O (LLO) in cellular immunity to Listeria monocytogenes. A correlation between LLO production by infecting bacteria and generation of protective immunity to virulent LLO-producing bacteria was noted. Using isogeneic hemolysin (Hly+ or Hly-) strains of L. monocytogenes, we demonstrated that LLO production by infecting bacteria is required to elicit T cells reactive both to bacteria-associated Ag and to the secreted LLO molecule as measured by IL-2 production in vitro. Distinct sets of T cells specific for largely nonoverlapping pools of antigenic determinants represented by LLO and cell-associated Ag (heat-killed L. monocytogenes) are generated after infection. We have used models for prediction of T cell epitopes based on primary structure of LLO, and synthetic amphipathic LLO peptides were evaluated as Ag in vitro or as immunogenes in vivo. Infection of several strains of mice (H-2k and H-2d) with LLO-producing L. monocytogenes resulted in the generation of T cells that could respond consistently to two peptides, LLO 215-234 and LLO 354-371. Mouse strains lacking expression of I-E molecules (e.g., B10.A(4R) and C57BL/6) responded to LLO but not to the peptides tested. With C3HeB/FeJ mice, antibodies to I-Ek blocked the presentation of LLO 215-234. The importance of the N-terminal portion of LLO 215-234 was evidenced by the drastic reduction in antigenic activity of truncated peptides (e.g., LLO 221-234 and LLO 224-234). LLO 215-234, the strongest and most consistent activator of T cells from L. monocytogenes-immune mice, fit well some models for antigenic peptides in several ways. It could be predicted to form an amphipathic alpha-helix, it contained multiple "Rothbard motifs" (charged residue or glycine, two or three hydrophobic amino acids and then a glycine or polar residue), it had a net charge of +2, and it contained the correct spacing of amino acids (five to six residues between a hydrophobic and basic amino acid) that is characteristic of I-Ek-binding peptides. Immunization with 8 of 10 synthetic LLO peptides generated T cells that recognized the immunizing peptide in vitro, but such T cells were only poorly reactive with LLO. Our results indicate that LLO is an important target Ag for stimulation of CD4+ L. monocytogenes-specific T cells, and that LLO 215-234 is antigenically dominant in C3HeB/FeJ mice.