Differential interaction of synthetic peptides from the carboxyl-terminal regulatory domain of tubulin with microtubule-associated proteins.

In previous studies we have demonstrated that a 4-kd tubulin fragment, including amino acid residues from Phe418 to Glu450 in alpha-subunit and Phe408-Ala445 of the beta-sequence, plays a major role in controlling tubulin interactions leading to microtubule assembly. The 4-kd carboxyl-terminal domain also constitutes an essential domain for the interaction of microtubule-associated proteins (MAPs). Removal of the 4-kd fragment facilitates tubulin self-association and renders the assembly MAP-independent. In order to define the substructure of the tubulin domain for MAP interaction, we have examined the binding of 3H-acetylated C-terminal peptides to MAP-2 and tau. Two synthetic peptides from the low-homology region within the 4-kd domain alpha (430-441) and beta (422-434) and the peptide, alpha (401-410) of the high-homology region adjacent to the 4-kd domain, were analyzed with respect to MAP interaction. The binding data showed a relatively strong interaction of MAP-2 with the beta (422-434) peptide and a weaker interaction of both MAPs components with alpha (430-441) tubulin peptide as analyzed by Airfuge ultracentrifugation and zone filtration chromatography. The homologous alpha (401-410) peptide did not bind to either MAP-2 or tau. Equilibrium dialysis experiments showed a co-operative binding of beta (422-434) peptide to multiple sites in tau. The alpha (430-441) peptide exhibited a stronger interaction for tau as compared with MAP-2.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of recurrent hydrophobic residues in catalysis of helix formation by T cell-presented peptides in the presence of lipid vesicles

We tested the hypothesis that the recurrence of hydrophobic amino acids in a polypeptide at positions falling in an axial, hydrophobic strip if the sequence were coiled as an alpha helix, can lead to helical nucleation on a hydrophobic surface. The hydrophobic surface could anchor such residues, whereas the peptide sequence grows in a helical configuration that is stabilized by hydrogen bonds among carbonyl and amido NH groups along the peptidyl backbone of the helix, and by other intercycle interactions among amino acid side chains. Such bound, helical structures might protect peptides from proteases and/or facilitate transport to a MHC-containing compartment and thus be reflected in the selection of T cell-presented segments. Helical structure in a series of HPLC-purified peptides was estimated from circular dichroism measurements in: 1) 0.01 M phosphate buffer, pH 7.0, 2) that buffer with 45% trifluoroethanol (TFE), and 3) that buffer with di-O-hexadecyl phosphatidylcholine vesicles. By decreasing the dielectric constant of the buffer, TFE enhances intrapeptide interactions generally, whereas the lipid vesicles only provide a surface for hydrophobic interactions. The peptides varied in their strip-of-helix hydrophobicity indices (SOHHI; the mean Kyte-Doolittle hydrophobicities of residues in an axial strip of an alpha helix) and in proline content. Structural order for peptides with helical circular dichroism spectra was estimated as percentage helicity from circular dichroism theta 222 nm values and peptide concentration. A prototypic alpha helical peptide with three cycles plus two amino acids and an axial hydrophobic strip of four leucyl residues (SOHHI = 3.8) was disordered in phosphate buffer, 58% helical in that buffer with 48% TFE, and 36% helical in that buffer with vesicles. Percentage helicity in the presence of vesicles of the subset of peptides without proline followed their SOHHI values. Peptides with multiple prolyl residues had circular dichroism spectra with strong signals, but since they did not have altered spectra in the presence of vesicles relative to phosphate buffer alone, the hydrophobic surface of the vesicle did not appear to stabilize those structures.     

Chemical synthesis of five tubulin antigenic sequences. Production and characterization of their corresponding anti-tubulin monospecific antibodies

Five peptides comprising several potential epitopes of alpha and beta-tubulin were synthesized by solid phase methods and purified to homogeneity by HPLC. These are RRNLDIERPTYTN (corresponding to positions 214-226 of the alpha chain of porcine brain tubulin), KDYEEVGVDSVEGE (alpha, 430-443), EGEFSEAREDMAALEKDYEEVGVDSVEGE (alpha, 415-443), RYPGQLNADLRKLAVN (beta, 241-256) and ESNMNDLVSEYQQYQDATAD (beta, 412-431). Appropriate conjugates with carrier proteins rendered all peptides immunogenic, raising antibodies that were cross-reactive with plate-adsorbed tubulin in ELISA. This reaction was completely inhibited by the corresponding free peptides, which indicates that these antibodies react specifically with the regions of alpha and beta-tubulin encompassed by the peptides. The reaction was also fully inhibited by soluble tubulin in a stabilising buffer. In immunoblotting experiments, the anti-peptide antibodies were useful as specific markers for either alpha- or beta-tubulin in brain extracts. The specificity of the anti-peptide antibodies for cellular microtubules was also examined by indirect immunofluorescence experiments.     

Tubulin structure probed with antibodies to synthetic peptides. Mapping of three major types of limited proteolysis fragments

We synthesized five peptides homologous to the potentially antigenic positions alpha(214-226), alpha(430-443), alpha(415-443), beta(241-256), and beta(412-431) of the porcine brain tubulin sequences. These peptides were successfully employed to raise tubulin-cross-reactive antibodies. The antibodies are specific of the regions of tubulin spanned by the peptides. They react specifically with the tubulin bands in immunoblots and with microtubules in immunofluorescence assays of cytoskeletons. The peptides of the C-terminal regions have also been employed to localize determinants recognized by two available monoclonal antibodies to tubulin in the positions alpha(415-430) and beta(412-431), respectively. In a first application of the anti-peptide antibodies, we have mapped the fragments of limited proteolysis of purified calf brain tubulin by trypsin, chymotrypsin, papain, thermolysin, subtilisin, and protease V8 from Staphylococcus aureus. Thirty-seven peptides have been identified, of which 32 have been unequivocally aligned into the tubulin sequences on the basis of their antigenic reactivity. There are three major, well-defined zones of preferential cleavage by the proteases: the C-termini and two internal zones in each chain. C-Terminal cleavages of both chains by subtilisin do not remove the antigenic reactivity of the zones alpha(415-430) and beta(412-431). C-Terminal cleavages by protease V8 are preferential of beta-tubulin. All six proteases tested cleave alpha- and/or beta-tubulin at one or both of the internal zones. These zones are located roughly at one-third and two-thirds of the chain length in both subunits. Therefore, a model of the tubulin monomers is proposed which consists of three major, proteolytically defined, compact regions (N-terminal, middle, and C-terminal thirds) and the cleavable zones. This model is discussed with the tubulin structural information presently available.     

Persistence of the alpha-helix stop signal in the S-peptide in trifluoroethanol solutions

alpha-Helix formation in the S-peptide (residues 1-19 of ribonuclease A) was studied in detail by use of two-dimensional 1H nuclear magnetic resonance to monitor the effects of 2,2,2-trifluoroethanol (TFE) at 0 degrees C and pH* 2.07. TFE stabilizes the S-peptide alpha-helix. Helix formation by a particular amino acid was monitored by the chemical shifts of the C alpha, C beta, and C gamma protons while increasing the concentration of TFE: large changes in chemical shift of a particular residue indicate that it is induced to go helical, whereas small chemical shift changes indicate little helix formation. Residues Thr-3 to Met-13 undergo chemical shift changes consistent with helix formation, whereas the other residues do not. Earlier work [Kim, P. S., & Baldwin, R. L. (1984) Nature 307, 329-334] reported that residues Thr-3 to His-12 become helical in aqueous solution. The existence of a "helix stop signal" was inferred from this behavior. We thus conclude that this helix stop signal persists in TFE solutions.     

Conformational exchange on the microsecond time scale in alpha-helix and beta-hairpin peptides measured by 13C NMR transverse relaxation

13C-NMR relaxation experiments (T(1), T(2), T(1)(rho), and NOE) were performed on selectively enriched residues in two peptides, one hydrophobic staple alpha-helix-forming peptide GFSKAELAKARAAKRGGY and one beta-hairpin-forming peptide RGITVNGKTYGR, in water and in water/trifluoroethanol (TFE). Exchange contributions, R(ex), to spin-spin relaxation rates for (13)C(alpha) and (13)C(beta) groups were derived and were ascribed to be mainly due to peptide folding-unfolding. To evaluate the exchange time, tau(ex), from R(ex), the chemical shift difference between folded and unfolded states, Deltadelta, and the populations of these states, p(i), were determined from the temperature dependence of (13)C chemical shifts. For both peptides, values for tau(ex) fell in the 1 micros to 10 micros range. Under conditions where the peptides are most folded (water/TFE, 5 degrees C), tau(ex) values for all residues in each respective peptide were essentially the same, supporting the presence of a global folding-unfolding exchange process. Rounded-up average tau(ex) values were 4 micros for the helix peptide and 9 micros for the hairpin peptide. This 2-3-fold difference in exchange times between helix and hairpin peptides is consistent with that observed for folding-unfolding of other small peptides.     

Two peptide fragments G55-I72 and K97-A109 from staphylococcal nuclease exhibit different behaviors in conformational preferences for helix formation

Two synthetic peptides, SNasealpha1 and SNasealpha2, corresponding to residues G55-I72 and K97-A109, respectively, of staphylococcal nuclease (SNase), are adopted for detecting the role of helix alpha1 (E57-A69) and helix alpha2 (M98-Q106) in the initiation of folding of SNase. The helix-forming tendencies of the two SNase peptide fragments are investigated using circular dichroism (CD) and two-dimensional (2D) nuclear magnetic resonance (NMR) methods in water and 40% trifluoroethanol (TFE) solutions. The coil-helix conformational transitions of the two peptides in the TFE-H2O mixture are different from each other. SNasealpha1 adopts a low population of localized helical conformation in water, and shows a gradual transition to helical conformation with increasing concentrations of TFE. SNasealpha2 is essentially unstructured in water, but undergoes a cooperative transition to a predominantly helical conformation at high TFE concentrations. Using the NMR data obtained in the presence of 40% TFE, an ensemble of alpha-helical structures has been calculated for both peptides in the absence of tertiary interactions. Analysis of all the experimental data available indicates that formation of ordered alpha-helical structures in the segments E57-A69 and M98-Q106 of SNase may require nonlocal interactions through transient contact with hydrophobic residues in other parts of the protein to stabilize the helical conformations in the folding. The folding of helix alpha1 is supposed to be effective in initiating protein folding. The formation of helix alpha2 depends strongly on the hydrophobic environment created in the protein folding, and is more important in the stabilization of the tertiary conformation of SNase.     

Structure-function analysis of Arg-Gly-Asp helix motifs in alpha v beta 6 integrin ligands

Data relating to the structural basis of ligand recognition by integrins are limited. Here we describe the physical requirements for high affinity binding of ligands to alpha v beta6. By combining a series of structural analyses with functional testing, we show that 20-mer peptide ligands, derived from high affinity ligands of alpha v beta6 (foot-and-mouth-disease virus, latency associated peptide), have a common structure comprising an Arg-Gly-Asp motif at the tip of a hairpin turn followed immediately by a C-terminal helix. This arrangement allows two conserved Leu/Ile residues at Asp(+1) and Asp(+4) to be presented on the outside face of the helix enabling a potential hydrophobic interaction with the alpha v beta6 integrin, in addition to the Arg-Gly-Asp interaction. The extent of the helix determines peptide affinity for alpha v beta6 and potency as an alpha v beta6 antagonist. A major role of this C-terminal helix is likely to be the correct positioning of the Asp(+1) and Asp(+4) residues. These data suggest an explanation for several biological functions of alpha v beta6 and provide a structural platform for design of alpha v beta6 antagonists.     

Helix formation in reduced,S-carboxymethylated human choriogonadotropin β subunit and tryptic peptides

The β subunit of human choriogonadotropin (hCGβ) and its asialoderivative were digested with trypsin and then reduced and S-carboxymethylated. A series of peptides were purified which corresponded to residues 1–43, 44–95, 96–114, and 123–145 of the 145 amino acid residue glycoprotein. The two N-linked oligosaccharides were present on the amino terminal peptide, and three of the four O-linked oligosaccharides were present on the carboxy terminal peptide. Circular dichroic spectra between 190–240 nm were obtained on reduced, S-carboxymethylated (RCM) hCGβ and the above peptides, both in aqueous solution and in the helicogenic solvent 80% (vol/vol) trifluoroethanol (TFE). In aqueous solution there was evidence of only limited helicity in the peptides and RCM-hCGβ however, in the presence of TFE, peptides 1–43 and 44–95 exhibited significant helicity, as did the full-length linear chain. The helicity developed in TFE by RCM-hCGβ appears much greater than that which occurs in the native, disulfide-intact form, thus suggesting that the disulfides prevent expression of helicity in regions with α-helix potential. Application of the Chou-Fasman secondary structure predictive algorithm to hCGβ suggested that several regions of helix potential, in particular regions 14–21, 59–69, and perhaps 80–88, may account for much of the helicity observed in peptides 1–43 and 44–95, respectively, in TFE. The region from 96–145 has no significant potential for helicity, consistent with the measured circular dichroic spectra of peptides 96–114 and 123–145. These results demonstrate that helicity can occur in the linear form of hCGβ, and this secondary structure can best be attributed to the amino terminal and the middle portion of the molecule. Several potential regions of β-structure and β-turns were also suggested.     

Tubulin assembly probed with antibodies to synthetic peptides.

Antibodies to synthetic peptides from the alpha and beta-tubulin sequences were employed to study zones of this protein active in microtubule assembly. In purified calf brain tubulin, six short sequences, selected according to their hydrophilicity and conservation, were found to be accessible to their affinity-purified immunoglobulin G (IgG) antibodies, in a competition radioimmunoassay performed under non-assembly native conditions. This indicated that the six sequences are exposed on the surface of the tubulin alpha beta heterodimer. IgG antibodies to the alpha(430-443) and beta(412-431) sequences perturbed substoichiometrically the assembly of purified tubulin, inducing microtubule bundling and the formation of opened up structures. These positions, which are close to the C termini, were accessible to the anti-peptide antibodies in taxol-induced microtubules, Zn2(+)-induced tubulin sheets, Mg2(+)-induced tubulin rings and in PtK2 cell microtubules. This, together with the comparison of the sizes and gross shapes of the antibody probes and microtubules, suggested that these sequences might be located at the protruding parts of the protofilaments. Antibodies to positions alpha(155-168) did not react with microtubules, while the equivalent zone beta(153-165) was accessible. The alpha(214-226) and beta(241-256) sequences were antigenically occluded in the taxol microtubules, Zn2(+)-induced sheets and Mg2(+)-induced ring arrays, as well as in native microtubules from PtK2 cells, though they became reactive by fixation. This result strongly suggested that these two zones are close to tubulin-tubulin contact sites. A working model is proposed in which the positions alpha(214-226) and beta(241-256) are close to the axial contacts between heterodimers, which lead to protofilament formation, while the positions alpha(241-256) and beta(214-226) are suggested to be related to the alpha-beta binding interface within the heterodimer.     

Tau induces ring and microtubule formation from alphabeta-tubulin dimers under nonassembly conditions

Tau is a neuronal microtubule-associated protein that plays a central role in many cellular processes, both physiological and pathological, such as axons stabilization and Alzheimer's disease. Despite extensive studies, very little is known about the detailed molecular basis of tau binding to microtubules. We used the four-repeat recombinant htau40 and tubulin dimers to show for the first time that tau is able to induce both microtubule and ring formation from 6S alphabeta tubulin in phosphate buffer without added magnesium (nonassembly conditions). The amount of microtubules or rings formed was protein concentration-, temperature-, and nucleotide-dependent. By means of biophysical approaches, we showed that tau binds to tubulin without global-folding change, detectable by circular dichroism. We also demonstrated that the tau-tubulin interaction follows a ligand-mediated elongation process, with two tau-binding site per tubulin dimer. Moreover, using a tubulin recombinant alpha-tubulin C-terminal fragment (404-451) and a beta-tubulin C-terminal fragment (394-445), we demonstrated the involvement of both of these tubulin regions in tau binding. From this model system, we gain new insight into the mechanisms by which tau binds to tubulin and induces microtubule formation.     

Carboxy-terminal regions on the surface of tubulin and microtubules. Epitope locations of YOL1/34, DM1A and DM1B

Tryptic and cyanogen bromide peptides of pig brain alpha- and beta-tubulin reacting with monoclonal antibodies YOL1/34, DM1A and DM1B have been isolated and identified. They all correspond to parts of the C-terminal regions of either alpha- or beta-tubulin, and those peptides reacting with a given antibody have overlapping sequences. In the case of YOL1/34, its relatively high reactivity with small peptides suggests that many of the determinants for this antibody are within the overlapping region of these peptides comprising only nine amino acids in positions alpha 414 to 422. The smallest common region of peptides reacting with the other alpha-tubulin antibody DM1A corresponds to positions alpha 426 to 450, whereby amino acids within the positions 426 and 430 appear to be particularly important for reactivity. Since the last C-terminal residues of alpha-tubulin are also accessible to antibodies and enzymes, it seems that an extensive part (35 to 40 residues) of this very acidic C-terminal domain is exposed on the surface of native tubulin dimers. In microtubules, however, the amino-terminal end of this region appears to be less accessible, as YOL1/34 reacts poorly, if at all, with intact microtubules. All of the peptides reacting with beta-tubulin monoclonal antibody DM1B were derived from the acidic C-terminal domain and they overlapped in positions beta 416 to 430. This indicates that beta-tubulin is also positioned with at least part of its acidic C-terminal domain on the surface of microtubules, since DM1B reacts with unfixed microtubules after microinjection.     

Amyloid fibril formation by peptide LYS (11-36) in aqueous trifluoroethanol

Peptide LYS (11-36), derived from the beta-sheet region of T4 lysozyme, forms an amyloid fibril in aqueous trifluoroethanol (TFE) at elevated temperature. The peptide has a moderate alpha-helix content in 20 and 50% (v/v) TFE solution; large quantities of fibrils were formed after incubation at 55 degrees C for 2 weeks as monitored by a thioflavin T fluorescence assay. No fibrils were observed when the peptide initially existed predominantly as a random coil or as a complete alpha helix. Our results suggest that a moderate amount of alpha helix and random coil present in the peptide initially facilitates the fibril-formation process, but a high alpha-helix content inhibits fibril formation. Transmission electron microscopy revealed several types of fibril morphologies at different TFE concentrations. The fibrils were highly twisted and consisted of interleaved protofilaments in 50% TFE, while smooth and flat ribbonlike fibrils were found in 20% TFE. In 50% TFE, the fibril growth rate of LYS (11-36) was found to depend strongly on peptide concentration and seeding but was insensitive to solution pH and ionic strength.     

HLA-B2702 (77-83/83-77) peptide binds to beta-tubulin on human NK cells and blocks their cytotoxic capacity

It has been described that peptides derived from a highly conserved region of the alpha1 helix of the first domain of HLA class I Ags exhibit immunomodulatory capacity blocking both T and NK cell cytotoxicity. In vivo treatment with these peptides prolongs survival of MHC-mismatched allografts. However, the molecular bases of these effects are still unclear. In this study, we further analyze the mechanisms by which the dimeric peptide HLA-B2702 (77-83/83-77) induces suppression of NK cell cytotoxicity. This peptide inhibits natural and redirected lysis mediated by NK cells without significantly affecting effector-target cell binding. We have also isolated and sequenced a protein that binds this inhibitory peptide, which structurally corresponds to beta-tubulin. Tubulin is the major protein of microtubules and is involved in target cell killing. Furthermore, B2702 peptide promotes GTP-independent tubulin assembly, producing aggregates that cannot be depolymerized by cold. Treatment of NK cells with Taxol or demecolcine, which interfere with microtubule organization, also prevents NK cell cytotoxicity. Taken together, these results support the hypothesis that the peptide B2702 (77-83/83-77) exerts its inhibitory effect on NK cell cytotoxicity by inducing polymerization of microtubules and interfering with their normal assembly/disassembly dynamics.     

Comparative structure analysis of tyrosine and valine residues in unprocessed silk fibroin (silk I) and in the processed silk fiber (silk II) from Bombyx mori using solid-state (13)C,(15)N, and (2)H NMR

The solid-state (13)C CP-MAS NMR spectra of biosynthetically labeled [(13)C(alpha)]Tyr, [(13)C(beta)]Tyr, and [(13)C(alpha)]Val silk fibroin samples of Bombyx mori, in silk I (the solid-state structure before spinning) and silk II (the solid-state structure after spinning) forms, have been examined to gain insight into the conformational preferences of the semicrystalline regions. To establish the relationship between the primary structure of B. mori silk fibroin and the "local" structure, the conformation-dependent (13)C chemical shift contour plots for Tyr C(alpha), Tyr C(beta), and Val C(alpha) carbons were generated from the atomic coordinates of high-resolution crystal structures of 40 proteins and their characteristic (13)C isotropic NMR chemical shifts. From comparison of the observed Tyr C(alpha) and Tyr C(beta) chemical shifts with those predicted by the contour plots, there is strong evidence in favor of an antiparallel beta-sheet structure of the Tyr residues in the silk fibroin fibers. On the other hand, Tyr residues take a random coil conformation in the fibroin film with a silk I form. The Val residues are likely to assume a structure similar to those of Tyr residues in silk fiber and film. Solid-state (2)H NMR measurements of [3,3-(2)H(2)]Tyr-labeled B. mori silk fibroin indicate that the local mobility of the backbone and the C(alpha)-C(beta) bond is essentially "static" in both silk I and silk II forms. The orientation-dependent (i.e., parallel and perpendicular to the magnetic field) solid-state (15)N NMR spectra of biosynthetically labeled [(15)N]Tyr and [(15)N]Val silk fibers reveal the presence of highly oriented semicrystalline regions.     

Retinal cyclic-GMP phosphodiesterase gamma-subunit: identification of functional residues in the inhibitory region.

Previously, we have domain-mapped the 87 amino acid PDE gamma inhibitory subunit of the retinal phosphodiesterase (PDE) alpha beta gamma 2 complex using synthetic peptides. The PDE gamma subunit has a binding domain for transducin-alpha (T alpha) and for PDE alpha/beta within residues # 24-45 and an inhibitory region for PDE alpha/beta within residues # 80-87. In order to establish the role of individual amino acids in the function of the PDE gamma inhibitory subunit, peptides of PDE gamma # 63-87 and mutant peptides were synthesized and utilized in PDE inhibition assays. The following peptides exhibited a decreased ability to inhibit PDE alpha/beta: All were from PDE gamma # 63-87; PDE gamma Tyr 84----Gly, PDE gamma Phe 73----Gly and PDE gamma Gln 83----Gly.     

Conformational studies of parathyroid hormone (PTH)/PTH-related protein (PTHrp) chimeric peptides

The N-terminal 1-34 segments of both parathyroid hormone (PTH) and parathyroid hormone-related protein (PTHrP) bind and activate the same membrane-embedded G protein-coupled receptor (PTH1 Rc) present on the surface of cells in target tissues such as bone and kidney. This binding occurs in spite of major differences between the two hormones in their amino acid sequence. Recently, it was shown that in (1-34) PTH/PTHrP hybrid peptides, the N-terminal 1-14 segment of PTHrP is incompatible with the C-terminal 15-34 region of PTH in terms of bioactivity. The sites of incompatibility were identified at positions 5 in PTHrP and 19 in PTH. In the present paper we describe the synthesis, biological evaluation, and conformational characterization of two segmental hybrids: PTHrP(1-27)-[Tyr(34)]bPTH(28-34)-NH(2) (hybrid I) and PTHrP(1-18)-[Nal(23), Tyr(34)]bPTH(19-34)-NH(2) (hybrid II). Hybrid I is as active as PTH(1-34)NH(2) and more than two orders of magnitude more active than hybrid II. The conformational properties of the hybrids were studied in water/trifluoroethanol (TFE) mixtures and in aqueous solutions containing dodecylphosphocholine (DPC) micelles by CD, two-dimensional nmr and computer simulations. Upon addition of TFE to the aqueous solution, both hybrids undergo a coil-helix transition. The helix content in 1:1 water/TFE obtained by CD data is about 75% for both hybrids. In the presence of DPC, helix formation is observed at detergent concentrations above critical micellar concentration and the maximum helix content is of approximately 35 and approximately 30% for hybrid I and II, respectively. Combined nmr analysis, distance geometry, and molecular dynamics calculations suggest that, in both solvent systems, the biologically active hybrid I exhibits two flexible sites, centered at residues 12 and 19, connecting helical segments. The flexibility point at position 19 is not present in the poorly active hybrid II. Our findings support the hypothesis, proposed in our previous work, that in bioactive PTH analogues the presence and location of flexibility points between helical segments are essential for enabling them to fold into the bioactive conformation upon interaction with the PTH1 receptor.     

Amphipathic control of the 3(10)-/alpha-helix equilibrium in synthetic peptides.

A series of short, amphipathic peptides incorporating 80% C(alpha),C(alpha)-disubstituted glycines has been prepared to investigate amphipathicity as a helix-stabilizing effect. The peptides were designed to adopt 3(10)- or alpha-helices based on amphipathic design of the primary sequence. Characterization by circular dichroism spectroscopy in various media (1 : 1 acetonitrile/water; 9 : 1 acetonitrile/water; 9 : 1 acetonitrile/TFE; 25 mM SDS micelles in water) indicates that the peptides selectively adopt their designed conformation in micellar environments. We speculate that steric effects from ith and ith + 3 residues interactions may destabilize the 3(10)-helix in peptides containing amino acids with large side-chains, as with 1-aminocyclohexane-1-carboxylic acid (Ac(6)c). This problem may be overcome by alternating large and small amino acids in the ith and ith + 3 residues, which are staggered in the 3(10)-helix.     

Conformational properties of the beta(400-436) and beta(400-445) C-terminal peptides of porcine brain tubulin.

Two peptides from the C-terminal region of the major beta-tubulin isotype (400-436 and 400-445) that include the critical areas for interaction with MAP2 and tau were examined to determine their conformations in aqueous solution. Despite a high theoretical potential for alpha-helix formation, CD spectroscopy showed that these peptides consisted primarily of random coil with some reverse turn. This was unaffected by the presence of counterions to the negatively charged side chains (Ca2+, Mg2+), but did change when the side-chain charges were neutralized by lowering the pH; under these conditions, the alpha-helix content of the longer peptide rose to 25% and the C-terminal truncated peptide to 15%. The peptides also adopt alpha-helical structure in the presence of trifluoroethanol, the truncated peptide again attaining a lower maximum percentage. The beta(400-445) peptide was also studied by 1-D and 2-D NMR techniques. The results indicate that at pH 5.6 or 7 in an aqueous solution the peptide is extremely flexible and lacks regular secondary structure, consistent with the CD results. Both peptides inhibited microtubule-associated protein-stimulated tubulin assembly, with the longer peptide being about 4 times as inhibitory as the smaller peptide. Neither was inhibitory in the absence of microtubule-associated proteins, indicating that interaction with this species was necessary for inhibition. The greater activity of the longer peptide could be due to the extra negative charges in this peptide and/or the greater tendency of this peptide to form an alpha-helical structure under the appropriate conditions.     

Spectroscopic evidence for backbone desolvation of helical peptides by 2,2,2-trifluoroethanol: an isotope-edited FTIR study

2,2,2-Trifluoroethanol (TFE) is widely used to induce helix formation in peptides and proteins, but the mechanism behind this effect is still poorly understood. Several recent papers have proposed that TFE acts by selectively desolvating the peptide backbone groups of the helix state. Infrared (IR) spectroscopy of the amide I band of polypeptides can be used to probe both secondary structure and backbone solvation, making this technique well suited for addressing the effect of TFE on polypeptide conformation. In this paper, we report the IR spectra as a function of TFE concentration for an alanine-rich peptide based on the repeat (AAKAA)(n)(). The IR spectra confirm that TFE desolvates the helical state of the peptide to a greater extent than the random coil state. Moreover, using a series of specifically (13)C-labeled peptides, the precise residues desolvated in the presence of TFE were identified. The residues most desolvated by TFE are the alanines located at position i - 4 in the sequence, where i is a lysine residue. This pattern of desolvation is consistent with molecular dynamics simulations which predict strong interactions between the lysine side chain at position n and the backbone carbonyl of the alanine at position i - 4. This is the first direct spectroscopic evidence of specific desolvation of helix backbone atoms in model alanine-rich peptides.