CD Spectra Of Peptides Research Articles

Characterization of Changes due to pH Variations in Beta Peptide (25–35) Leading to Alzheimer’s Disease

The effect of various concentrations of amyloid beta peptide (ABP) in different pH (pH 2, 6, 7, 8, 10) in aging at different time intervals was analysed using various techniques. The pH 7.4 and 8 were compared with other pH solution which indicated that the secondary structure content with high β-sheet conformation is found at pH 7.4 and 8. Phosphate buffer solutions (pH) were detected using CD spectroscopy. The CD spectra of peptide in phosphate buffer (pH 2 and 10) on aging at time intervals were also analysed with the aging of sample (pH 2) on the 10th day. The results showed no major secondary structural changes in the peptide, which was indicative predominant random coil structure. The phosphate buffer pH 6, 7.4, 8 on the 10th day showed the occurrence of crossover point. This same sample smear on slide was mounded and the fibrils were visualized using the light microscope. The percent of viable cell in different pH was detected in MTT (3-(4,5-Dimethyazol-2-yl)-2, 5-diphenyl-tetrazolium bromide) assay using human keratinocyte cell line (HaCaT). Fluorescence spectroscopy was used to study the growth and development of fibrils.

Bicyclic Peptide Based Lectinomimic

Peptide based lectin mimetics represent an attractive approach for the development of artificial carbohydrate receptors that might find application in bio-analytical and medicinal fields. Taking into consideration the structure of typical lectin binding site, we have designed a novel artificial receptor molecule possessing a rigid three-dimensional structure, hydrogen-bonding site and lipophilic binding pocket to promote hydrophobic interaction and hydrogen-bonding. A new solid- phase synthetic approach that allows complete synthesis of desired bicyclic peptide 1 on the solid support was developed. CD spectra of peptides 1 and 2 indicate that the structure of 1 is rather rigid and preorganised for the three-dimensional monosaccharide substrates binding. The binding affinities of bicyclic peptide receptor 1 toward various carbohydrate substrates at physiologically relevant conditions were estimated by UV/vis and fluorimetric titration experiments, and the observed values are in the millimolar range. With these results we have demonstrated that the bicyclic peptide 1 represent a promising basis for the design of new and more efficient carbohydrate receptors that may have broader application in bio-analytical or medicinal field.

A Synthetic S6 Segment Derived from KvAP Channel Self-assembles, Permeabilizes Lipid Vesicles, and Exhibits Ion Channel Activity in Bilayer Lipid Membrane

KvAP is a voltage-gated tetrameric K(+) channel with six transmembrane (S1-S6) segments in each monomer from the archaeon Aeropyrum pernix. The objective of the present investigation was to understand the plausible role of the S6 segment, which has been proposed to form the inner lining of the pore, in the membrane assembly and functional properties of KvAP channel. For this purpose, a 22-residue peptide, corresponding to the S6 transmembrane segment of KvAP (amino acids 218-239), and a scrambled peptide (S6-SCR) with rearrangement of only hydrophobic amino acids but without changing its composition were synthesized and characterized structurally and functionally. Although both peptides bound to the negatively charged phosphatidylcholine/phosphatidylglycerol model membrane with comparable affinity, significant differences were observed between these peptides in their localization, self-assembly, and aggregation properties onto this membrane. S6-SCR also exhibited reduced helical structures in SDS micelles and phosphatidylcholine/phosphatidylglycerol lipid vesicles as compared with the S6 peptide. Furthermore, the S6 peptide showed significant membrane-permeabilizing capability as evidenced by the release of calcein from the calcein-entrapped lipid vesicles, whereas S6-SCR showed much weaker efficacy. Interestingly, although the S6 peptide showed ion channel activity in the bilayer lipid membrane, despite having the same amino acid composition, S6-SCR was significantly inactive. The results demonstrated sequence-specific structural and functional properties of the S6 wild type peptide. The selected S6 segment is probably an important structural element that could play an important role in the membrane interaction, membrane assembly, and functional property of the KvAP channel.

Histone H4 Acetylation Differentially Modulates Arginine Methylation by an in Cis Mechanism

Histone H4 undergoes extensive post-translational modifications (PTMs) at its N-terminal tail. Many of these PTMs profoundly affect the on and off status of gene transcription. The molecular mechanism by which histone PTMs modulate genetic and epigenetic processes is not fully understood. In particular, how a PTM mark affects the presence and level of other histone modification marks needs to be addressed and is essential for better understanding the molecular basis of histone code hypothesis. To dissect the interplaying relationship between different histone modification marks, we investigated how individual lysine acetylations and their different combinations at the H4 tail affect Arg-3 methylation in cis. Our data reveal that the effect of lysine acetylation on arginine methylation depends on the site of acetylation and the type of methylation. Although certain acetylations present a repressive impact on PRMT1-mediated methylation (type I methylation), lysine acetylation generally is correlated with enhanced methylation by PRMT5 (type II dimethylation). In particular, Lys-5 acetylation decreases the activity of PRMT1 but increases that of PRMT5. Furthermore, circular dichroism study and computer simulation demonstrate that hyperacetylation increases the content of ordered secondary structures at the H4 tail region. These findings provide new insights into the regulatory mechanism of Arg-3 methylation by H4 acetylation and unravel the complex intercommunications that exist between different the PTM marks in cis. The divergent activities of PRMT1 and PRMT5 with respect to different acetyl-H4 substrates suggest that type I and type II protein-arginine methyltransferases use distinct molecular determinants for substrate recognition and catalysis.

Terminal effect of chiral residue on helical screw sense in achiral peptides

To understand the terminal effect of chiral residue for determining a helical screw sense, we adopted five kinds of peptides I–V containing N- and/or C-terminal chiral Leu residue(s): Boc–L-Leu–(Aib–ΔPhe)2–Aib–OMe (I), Boc–(Aib–ΔPhe)2–L-Leu–OMe (II), Boc–L-Leu–(Aib–ΔPhe)2–L-Leu–OMe (III), Boc–D-Leu–(Aib–ΔPhe)2–L-Leu–OMe (IV), and Boc–D-Leu–(Aib–ΔPhe)2–Aib–OMe (V). The segment –(Aib–ΔPhe)2– was used for a backbone composed of two “enantiomeric” (left-/right-handed) helices. Actually, this could be confirmed by 1H-nmr [nuclear Overhauser effect (NOE) and solvent accessibility of NH resonances] and CD spectroscopy on Boc–(Aib–ΔPhe)2–Aib–OMe, which took a left-/right-handed 310-helix. Peptides I–V were also found to take 310-type helical conformations in CDCl3, from difference NOE measurement and solvent accessibility of NH resonances. Chloroform, acetonitrile, methanol, and tetrahydrofuran were used for CD measurement. The CD spectra of peptides I–III in all solvents showed marked exciton couplets with a positive peak at longer wavelengths, indicating that their main chains prefer a left-handed screw sense over a right-handed one. Peptide V in all solvents showed exciton couplets with a negative peak at longer wavelengths, indicating it prefers a right-handed screw sense. Peptide IV in chloroform showed a nonsplit type CD pattern having only a small negative signal around 280 nm, meaning that left- and right-handed helices should exist with almost the same content. In the other solvents, peptide IV showed exciton couplets with a negative peak at longer wavelengths, corresponding to a right-handed screw sense. From conformational energy calculation and the above 1H-nmr studies, an N- or C-terminal L-Leu residue in the lowest energy left-handed 310-helical conformation was found to take an irregular conformation that deviates from a left-handed helix. The positional effect of the L-residue on helical screw sense was discussed based on CD data of peptides I–V and of Boc–(L-Leu–ΔPhe)n–L-Leu–OMe (n = 2 and 3). © 1999 John Wiley & Sons, Inc. Biopoly 49: 551–564, 1999

A Study of Chain-Length Effect on Helical Screw Sense in Peptides Having an N-Terminal L-Leu Residue

In order to clarify which helical screw sense is dominated depending on chain-length of peptides when an L-residue is introduced into the N-terminal position of achiral helical segments, we prepared three kinds of peptides I—III: Boc–L-Leu–(Aib-ΔPhe)n–Aib–OMe (n=2—4) I—III (Boc, t-butoxycarbonyl; Aib, α-aminoisobutyric acid; ΔPhe, Z-dehydrophenylalanine; OMe, methoxy). Here the segment –(Aib–ΔPhe)n–Aib–OMe was used for an achiral backbone composed of two “enantiomeric” (left-/right-handed) helices. Peptides I—III were found to form a 310-type helical conformation in chloroform, from the position of amide I band in FT-IR spectra and solvent accessibility of NH resonances in 1H NMR measurement. CD spectra of peptides I—III showed exciton couplets around 280 nm with a positive peak at longer wavelengths in chloroform, acetonitrile, methanol, and tetrahydrofuran. The sign of exciton couplets indicates that the main-chains prefer a left-handed screw sense. Consequently, an N-terminal L-Leu residue induces a left-handed screw sense preferentially, irrespective of chain lengths for achiral helical segments (5—9 residues).This result could be supported by conformational energy calculation on acetyl–L-Leu–(Aib–ΔPhe)4–Aib–OMe, in which a left-handed helical conformation was predominant. Also, in the lowest-energy left-handed helix, an N-terminal L-Leu residue takes an irregular conformation that deviates from a left-handed 310-/α-helical region in conformational space of (φ, ψ).

Enhanced antigenicity of a four-contact-residue epitope of the measles virus hemagglutinin protein by phage display libraries: evidence of a helical structure in the putative active site

Antigenicity and conformational propensities of synthetic peptides corresponding to the sequential epitope H236–255 of the measles virus hemagglutinin protein were investigated. This epitope corresponds to the neutralising and protective monoclonal antibody BH129 and includes Arg243, implicated in CD46-down-regulation and Arg253 that has been mapped to the putative enzymatic site. Fine mapping with truncation-, elongation-, Gly- and Ala-substitution analogues defined EL-QL as the critical residues of the minimal epitope S 244ELSQL 249. CD spectra of peptides, comparison with the 3D-structure of homologous sequences, and prediction algorithms suggested a helical structure with the contact residues E 245L–QL 249 located on the protein surface. Mimotopes obtained with a 6-mer phage display library contained a consensus Pro (important for binding) instead of Ser 247 of the wild-type sequence (irrelevant for binding). The kink induced by Pro seemed to be essential to bring the 4 contact-residues in the mimotopes and in the corresponding short peptides together. CD analysis and prediction algorithms suggested that non-helical conformations of the phage insert and of the peptides may favourably mimic the antigenic helical turns of the wild-type sequence, resulting in an up to 135 times higher antigenicity of the mAb towards the mimotope peptides.

A Set of Constructed Type Spectra for the Practical Estimation of Peptide Secondary Structure from Circular Dichroism

While reliable methods have been developed for the estimation of globular protein secondary structure content from their far UV circular dichroism spectra, these are not suitable for the analysis of simple peptides. Model peptides have been measured in purely α-helical, β-sheet, and coil form, and are often used for fitting the CD spectra of peptides, but these are mainly derived from homopolymers and exhibit side-chain-dependent characteristics that do not accurately represent the situation in natural sequences. We have attempted to reduce the side-chain bias inherent in these spectra by constructing a series of frequency-weighted “average” spectra from all available data on model peptides. These have proved quite satisfactory in estimating the secondary structure of a number of peptides. A computer program incorporating these spectra has been developed for practical use.

Correcting the Circular Dichroism Spectra of Peptides for Contributions of Absorbing Side Chains

The aromatic and sulfur-containing side chains Trp, Tyr, Phe, Cys, and Met contribute to the CD spectra of peptides and proteins in the amide region, interfering with the analysis for secondary structure. We propose a method to correct the CD spectra of peptides undergoing the helix–coil transition for contributions due to absorbing side chains using singular value decomposition. The method uses the common basis vectors obtained from an analysis of the CD spectra of related peptides without the aromatic and sulfur-containing amino acids. The common basis vectors are fitted to a portion of the CD spectrum of the peptide being corrected, in the range that is unaffected by its sidechain contributions. Then the resulting coefficients from the fitting are used along with the common basis vectors to regenerate the entire corrected spectrum. The method is illustrated for the CD spectra of the peptide sequence acetyl-Y-VAXAK-VAXAK-VAXAK-amide, where X is substituted with the 20 naturally occurring amino acids. This peptide model adopts a random-coil conformation in 2 mmsodium phosphate buffer, pH 5.5, and becomes an α helix in methanol/buffer solutions. The difference between the original and corrected spectra shows the contribution from the aromatic and sulfur-containing side chains.

Design of a Helical Backbone. Conformation of Sequential Tridecapeptide Containing Z-Dehydrophenylalanine Residues

Sequential peptides containing Z-dehydrophenylalanine (ΔZPhe) residues, Boc-(L-Leu-ΔZPhe)n-L-Leu-OMe (n=2–6), were synthesized, and their conformations in solution were investigated using 1H NMR and CD spectroscopy. Tridecapeptide (n=6) was shown to adopt a helical conformation in CDCl3 by observation of successive NiH↔Ni+1H nuclear Overhauser effects. Solvent accessibility of NH resonances for peptide n=6 in CDCl3 indicated that all the NH groups except those belonging to the N-terminal Leu-ΔZPhe moiety participate in intramolecular hydrogen bonding. Similar accessibility was observed for peptides n=2–5. Thus, peptides n=2–6 in CDCl3 show a (i+3)→i intramolecular hydrogen bonding pattern characteristic of a 310-helix. CD spectra of peptides n=3–6 in chloroform showed exciton couplets around 280 nm with a negative peak at longer wavelengths, corresponding to a right-handed helical sense. ΔZPhe residue was shown to be effective to design a 310-helical-type backbone using sequential peptidcs of (X-ΔZPhe) unit.

Biological and physical properties of a beta-endorphin analog containing only D-amino acids in the amphiphilic helical segment 13-31.

Our approach to the modeling of beta-endorphin has been based on the proposal that three basic structural units can be distinguished in the natural peptide hormone: a highly specific opiate recognition sequence at the N terminus (residues 1-5) connected via a hydrophilic link (residues 6-12) to a potential amphiphilic helix in the C-terminal residues 13-31. Our previous studies showed the validity of this approach and have demonstrated the importance of the amphiphilic helical structure in the C terminus of beta-endorphin. The present model, peptide 5, has been designed in order to evaluate further the requirements of the amphiphilic secondary structure as well as to determine the importance of this basic structural element as compared to more specific structural features which might occur in the C-terminal segment. For these reasons, peptide 5 retains the three structural units previously postulated for beta-endorphin; the major difference with regard to previous models is that the whole C-terminal segment, residues 13-31, has been built using only D-amino acids. In aqueous buffered solutions as well as in 2,2,2-trifluoroethanol-containing solutions, the CD spectra of peptide 5 show the presence of a considerable amount of left-handed helical structure. Enzymatic degradation studies employing rat brain homogenate indicate that peptide 5 is stable in this milieu. In delta- and mu-opiate receptor-binding assays, peptide 5 shows a slightly higher affinity than beta-endorphin for both receptors while retaining the same delta/mu selectivity. In opiate assays on the guinea pig ileum, the potency of peptide 5 is twice that of beta-endorphin. In the rat vas deferens assay, which is very specific for beta-endorphin, peptide 5 displays mixed agonist-antagonist activity. Most remarkably, peptide 5 displays a potent opiate analgesic effect when injected intracerebroventricularly into mice. At equal doses, the analgesic effect of peptide 5 is less than that of beta-endorphin (10-15%) but longer lasting. In conjunction with our previous model studies, these results clearly demonstrate that the amphiphilic helical structure in the C terminus of beta-endorphin is of predominant importance with regard to activity in rat vas deferens and analgesic assays. The similarity between the in vitro and in vivo opiate activities of beta-endorphin and peptide 5, when compared to the drastic change in chirality in the latter model, demonstrates that even a left-handed amphiphilic helix formed by D-amino acids can function satisfactorily as a structural unit in a beta-endorphin-like peptide.