C-terminal Amide Research Articles

Theoretical examination of competitive β-radical-induced cleavages of N–Cα and Cα–C bonds of peptides

Selective cleavages of N–Cα and Cα–C bonds of β-radical tautomers of amino acid residues in radical peptides have been examined theoretically by means of the density functional theory at the M06-2X/6-311++G(d,p) level. The majority of the bond cleavages are homolytic via β-scission. Their energy barriers depend largely on the ability of the radical being stabilized in the transition structures and the availability of a mobile proton in the vicinity of the β-radical center. The N–Cα bond is less favorably cleaved than the Cα–C bond (except Ser and Thr) for systems without a mobile proton. It is because, firstly, the homolytic cleavage is less favorable for the more polar N–Cα bond than for the less polar Cα–C bond. Secondly, a less stable σ-radical localized on the amide nitrogen atom of the incipient N-terminal fragment is formed for the former, while a more stable radical delocalized in a π*(CO)-like orbital of the incipient C-terminal fragment is formed for the latter. In the presence of a mobile proton N-terminal to the β-radical center, some degrees of heterolytic cleavage character, as preferred by the polar N–Cα bond, are observed. Consequently, its barrier is reduced. If the mobile proton is located at the C-terminal amide oxygen of the β-radical center, the Cα–C bond cleavage will be significantly suppressed. It is because the radical in the incipient C-terminal fragment becomes more localized as a σ-radical on the carbon atom of its protonated amide group. With basic amino acid residues, the Cα–C bond cleavage can be reactivated. Heterolytic cleavage of the polar N–Cα bond can be largely facilitated if a mobile proton N-terminal to the β-radical center is available and the radical in the incipient C-terminal fragment is sufficiently stabilized, for instance, by the aromatic side chain of Trp and Tyr. Therefore, cleavages of the N–Cα bond induced by the β-radical tautomer of Trp and Tyr are often preferred as compared with cleavages of the Cα–C bond in peptide radical cations containing mobile protons.

Expression of recombinant human bifunctional peptidylglycine α-amidating monooxygenase in CHO cells and its use for insulin analogue modification

The availability of catalytically active peptidylglycine α-amidating monooxygenase (PAM) should provide the means to examine its potential use for the chemienzymatic synthesis of bioactive peptides for the purpose of pharmacological studies. Hypoglycemic activity is one of the most important features of insulin derivatives. Insulin glargine amide was found to show a time/effect profile which is distinctly more flat and thus more advantageous than insulin glargine itself. The aim of the study was to obtain recombinant PAM and use it for insulin analogue amidation. We stably expressed a recombinant PAM in CHO dhfr-cells in culture. Recombinant PAM was partially purified by fractional ammonium sulphate precipitation and ion-exchange chromatography. The enzyme was used to modify glycine-extended A22(G)-B31(K)–B32(R) human insulin analogue (GKR). Alpha-amidated insulin was analyzed by HPLC and mass spectrometry. Hypoglycemic activity of amidated and non-amidated insulin was compared. The pharmacodynamic effect was based on glucose concentration measurement in Wistar rats with hyperglycemia induced by streptozotocin. The overall glycemic profile up to 36 h was evaluated after subcutaneous single dosing at a range of 2.5–7.5 U/kg b.w. The experiment on rats confirmed with a statistical significance (P < 0.05) hypoglycemic activity of GKR-NH2 in comparison to a control group receiving 0.9% NaCl. Characteristics for GKR-NH2 profile was a rather fast beginning of action (0.5–2.0 h) and quite prolonged return to initial values. GKR-NH2 is a candidate for a hypoglycemic drug product in diabetes care. In addition, this work also provides a valuable alternative method for preparing any other recombinant bioactive peptides with C-terminal amidation.

Controls and constrains of the membrane disrupting action of Aurein 1.2.

Aurein 1.2 is a 13 residue antimicrobial peptide secreted by the Australian tree frog Litoria Aurea. It is a surface-acting membrane disrupting peptide that permeabilizes bacterial membranes via the carpet mechanism; the molecular details of this process are mostly unknown. Here the mechanism of action of Aurein 1.2 was investigated with an emphasis on the role of membrane charge and C-terminal amidation of the peptide. Using quartz crystal microbalance (QCM) fingerprinting it was found that the membrane charge correlates with membrane affinity of the peptide, however the binding and the membrane disrupting processes are not charge driven; increased membrane charge reduces the membrane disrupting activity. Coarse grain simulations revealed that phenylalanine residues act as membrane anchors. Accordingly Aurein 1.2 has the ability to bind to any membrane. Furthermore, bundling precludes membrane disruption in case of wild type peptides, while non C-terminal amidated peptides form random aggregates leading to detachment from the membrane. Hence C-terminal amidation is crucial for Aurein 1.2 action. Our results suggest that Aurein 1.2 acts via aggregation driven membrane penetration. The concomitant change in the tension of the outer leaflet imposes a spontaneous curvature on the membrane, leading to disintegration.

The natural scorpion peptide, BmK NT1 activates voltage-gated sodium channels and produces neurotoxicity in primary cultured cerebellar granule cells

The scorpion Buthus martensii Karsch has been used in Traditional Chinese Medicine to treat neuronal diseases such as neuropathic pain, paralysis and epilepsy for thousands of years. Studies have demonstrated that scorpion venom is the primary active component. Although scorpion venom can effectively attenuate pain in the clinic, it also produces neurotoxic response. In this study, toxicity guided purification led to identify a mammalian toxin termed BmK NT1 comprising of 65 amino acid residues and an amidated C-terminus, a mature peptide encoded by the nucleotide sequence (GenBank No. AF464898). In contract to the recombinant product of the same nucleotide sequence, BmK AGAP, which displayed analgesic and anti-tumor effect, intravenous injection (i.v.) of BmK NT1 produced acute toxicity in mice with an LD50 value of 1.36 mg/kg. In primary cultured cerebellar granule cells, BmK NT1 produced a concentration-dependent cell death with an IC50 value of 0.65 μM (0.41–1.03 μM, 95% Confidence Intervals, 95% CI) which was abolished by TTX, a voltage-gated sodium channel (VGSC) blocker. We also demonstrated that BmK NT1 produced modest sodium influx in cerebellar granule cell cultures with an EC50 value of 2.19 μM (0.76–6.40 μM, 95% CI), an effect similar to VGSC agonist, veratridine. The sodium influx response was abolished by TTX suggesting that BmK NT1-induced sodium influx is solely through activation of VGSC. Considered these data together, we demonstrated that BmK NT1 activated VGSC and produced neurotoxicity in cerebellar granule cell cultures.

Generation of a novel monoclonal antibody that recognizes the alpha (α)-amidated isoform of a valine residue.

BackgroundAlpha (α)-amidation of peptides is a mechanism required for the conversion of prohormones into functional peptide sequences that display biological activities, receptor recognition and signal transduction on target cells. Alpha (α)-amidation occurs in almost all species and amino acids identified in nature. C-terminal valine amide neuropeptides constitute the smallest group of functional peptide compounds identified in neurosecretory structures in vertebrate and invertebrate species.MethodsThe α-amidated isoform of valine residue (Val-CONH2) was conjugated to KLH-protein carrier and used to immunize mice. Hyperimmune animals displaying high titers of valine amide antisera were used to generate stable hybridoma-secreting mAbs. Three productive hybridoma (P15A4, P17C11, and P18C5) were tested against peptides antigens containing both the C-terminal α-amidated (–CONH2) and free α-carboxylic acid (−COO−) isovariant of the valine residue.ResultsP18C5 mAb displayed the highest specificity and selectivity against C-terminal valine amidated peptide antigens in different immunoassays. P18C5 mAb-immunoreactivity exhibited a wide distribution along the neuroaxis of the rat brain, particularly in brain areas that did not cross-match with the neuronal distribution of known valine amide neuropeptides (α-MSH, adrenorphin, secretin, UCN1-2). These brain regions varied in the relative amount of putative novel valine amide peptide immunoreactive material (nmol/μg protein) estimated through a fmol-sensitive solid-phase radioimmunoassay (RIA) raised for P18C5 mAb.ConclusionsOur results demonstrate the versatility of a single mAb able to differentiate between two structural subdomains of a single amino acid. This mAb offers a wide spectrum of potential applications in research and medicine, whose uses may extend from a biological reagent (used to detect valine amidated peptide substances in fluids and tissues) to a detoxifying reagent (used to neutralize exogenous toxic amide peptide compounds) or as a specific immunoreagent in immunotherapy settings (used to reduce tumor growth and tumorigenesis) among many others.

A novel ranacyclin-like peptide with anti-platelet activity identified from skin secretions of the frog Amolops loloensis

Albeit many bioactive peptides have been reported from amphibian skins, no anti-platelet peptide has been identified till to date. Here, an anti-platelet peptide, namely Zongdian platelet inhibitor (ZDPI), with the molecular weight of 1798.6Da, was purified and characterized from skin secretions of the frog, Amolops loloensis. The amino acid sequence of ZDPI was determined as FRGCWLKNYSPRGCL-NH2 by combination methods of Edman degradation, mass spectrometry analysis and carboxypeptidase Y treatment revealing that it is composed of 15 amino acid residues with two cysteines formed an intra-molecular disulfide bridge and C-terminal amidation. cDNA encoding ZDPI precursor was cloned from skin cDNA library of A. loloensis. The precursor is composed of 63 amino acid (aa) residues including the predicted signal peptide (22aa), an acidic spacer peptide (19aa), and mature ZDPI. BLAST search indicates that ZDPI belongs to antimicrobial peptide family of ranacyclin, peptide leucine arginine or odorranain. It was found to inhibit ADP-induced platelet aggregation in a dose-dependent manner. At the concentration of 32μg/ml, ZDPI completely inhibited platelet aggregation induced by ADP. To the best of our knowledge, this is the first report about an anti-platelet peptide from amphibian skin secretions. Considering its strong inhibitory ability on platelets and simple structure, ZDPI might be an excellent candidate or template to develop anti-thrombosis agent. In addition, the discovery of anti-platelet peptide in the frog skin increases biological function spectrum of amphibian skin peptides.

Striking Oxygen Sensitivity of the Peptidylglycine α-Amidating Monooxygenase (PAM) in Neuroendocrine Cells

Interactions between biological pathways and molecular oxygen require robust mechanisms for detecting and responding to changes in cellular oxygen availability, to support oxygen homeostasis. Peptidylglycine α-amidating monooxygenase (PAM) catalyzes a two-step reaction resulting in the C-terminal amidation of peptides, a process important for their stability and biological activity. Here we show that in human, mouse, and insect cells, peptide amidation is exquisitely sensitive to hypoxia. Different amidation events on chromogranin A, and on peptides processed from proopiomelanocortin, manifest similar striking sensitivity to hypoxia in a range of neuroendocrine cells, being progressively inhibited from mild (7% O2) to severe (1% O2) hypoxia. In developing Drosophila melanogaster larvae, FMRF amidation in thoracic ventral (Tv) neurons is strikingly suppressed by hypoxia. Our findings have thus defined a novel monooxygenase-based oxygen sensing mechanism that has the capacity to signal changes in oxygen availability to peptidergic pathways.

Characterization of acidic and basic variants of IgG1 therapeutic monoclonal antibodies based on non-denaturing IEF fractionation.

Characterization of both the acidic and basic regions of imaged capillary isoelectric focusing (icIEF) profile of an IgG1 antibody was achieved through preparative immobilized pH gradient isoelectric focusing (IPG-IEF) fractionation. Recent attempts at using this method to fractionate charge variants of monoclonal antibodies (mAbs) have shown promising results, but identification of the chemical modifications in the variants was limited to the basic species. We have optimized the method to achieve enrichment of each variant across the icIEF profile of an IgG1 mAb. The fractionation was followed by extended characterization to elucidate the composition of the acidic, main, and basic species observed in the icIEF profile. Deamidation, sialylation, glycation, and fragmentation were identified as the main modifications contributing to acidic variants of the mAb while C-terminal lysine, C-terminal proline amidation, and uncyclized N-terminal glutamine were the major species contributing to the basic variants. This characterization allows a better understanding of the modifications that contribute to the charge variants observed by icIEF, facilitating the evaluation of impacts on product safety and efficacy.

The anti-inflammatory action of the analgesic kyotorphin neuropeptide derivatives: insights of a lipid-mediated mechanism.

Recently, a designed class of efficient analgesic drugs derived from an endogenous neuropeptide, kyotorphin (KTP, Tyr-Arg) combining C-terminal amidation (KTP-NH2) and N-terminal conjugation to ibuprofen (Ib), IbKTP-NH2, was developed. The Ib moiety is an enhancer of KTP-NH2 analgesic action. In the present study, we have tested the hypothesis that KTP-NH2 is an enhancer of the Ib anti-inflammatory action. Moreover, the impact of the IbKTP-NH2 conjugation on microcirculation was also evaluated by a unified approach based on intravital microscopy in the murine cremasteric muscle. Our data show that KTP-NH2 and conjugates do not cause damage on microcirculatory environment and efficiently decrease the number of leukocyte rolling induced by lipopolysaccharide (LPS). Isothermal titration calorimetry showed that the drugs bind to LPS directly thus contributing to LPS aggregation and subsequent elimination. In a parallel study, molecular dynamics simulations and NMR data showed that the IbKTP-NH2 tandem adopts a preferential "stretched" conformation in lipid bilayers and micelles, with the simulations indicating that the Ib moiety is anchored in the hydrophobic core, which explains the improved partition of IbKTP-NH2 to membranes and the permeability of lipid bilayers to this conjugate relative to KTP-NH2. The ability to bind glycolipids concomitant to the anchoring in the lipid membranes through the Ib residue explains the analgesic potency of IbKTP-NH2 given the enriched glycocalyx of the blood-brain barrier cells. Accumulation of IbKTP-NH2 in the membrane favors both direct permeation and local interaction with putative receptors as the location of the KTP-NH2 residue of IbKTP-NH2 and free KTP-NH2 in lipid membranes is the same.

Editorial: A Comparative Survey of the RF-Amide Peptide Superfamily.

The first member of the RF-amide peptide superfamily to be characterized, in 1977, was the cardioexcitatory peptide, FMRFamide, isolated from the ganglia of the clam Macrocallista nimbosa (1). Since then, a large number of such peptides, designated after their C-terminal arginine (R) and amidated phenylalanine (F) residues, have been identified in representative species of all major phyla. The discovery, 12 years ago, that the RF-amide peptide kisspeptin, acting via GPR54, was essential for the onset of puberty and reproduction, has been a major breakthrough in reproductive physiology (2–4). It has also put in front of the spotlights RF-amide peptides and has invigorated research on this superfamily of regulatory neuropeptides. The present Research Topic aims at illustrating major advances achieved, through comparative studies in (mammalian and non-mammalian) vertebrates and invertebrates, in the knowledge of RF-amide peptides in terms of evolutionary history and physiological significance.

Structure-based Mechanistic Insights into Terminal Amide Synthase in Nosiheptide-Represented Thiopeptides Biosynthesis.

Nosiheptide is a parent compound of thiopeptide family that exhibit potent activities against various bacterial pathogens. Its C-terminal amide formation is catalyzed by NosA, which is an unusual strategy for maturating certain thiopeptides by processing their precursor peptides featuring a serine extension. We here report the crystal structure of truncated NosA1-111 variant, revealing three key elements, including basic lysine 49 (K49), acidic glutamic acid 101 (E101) and flexible C-terminal loop NosA112-151, are crucial to the catalytic terminal amide formation in nosiheptide biosynthesis. The side-chain of residue K49 and the C-terminal loop fasten the substrate through hydrogen bonds and hydrophobic interactions. The side-chain of residue E101 enhances nucleophilic attack of H2O to the methyl imine intermediate, leading to Cα-N bond cleavage and nosiheptide maturation. The sequence alignment of NosA and its homologs NocA, PbtH, TpdK and BerI, and the enzymatic assay suggest that the mechanistic studies on NosA present an intriguing paradigm about how NosA family members function during thiopeptide biosynthesis.

Fragmentation reactions of an (n = 4–8) ions containing only alanine residues

A detailed study of the fragmentation reactions of an (n=4–8) ions containing only Ala residues has been carried out using collisional activation on an electrospray/QqToF instrument. Two distinct fragmentation pathways are observed. One, which we have called the “imine” pathway, involves initial elimination of an imine (an→bn−1) followed by elimination of Ala residues (bn−1→bn−2, etc.). This pathway most likely involves the protonated substituted imine R2C(O)NH+CHR1. The second pathway, which we have called the “amide” pathway, involves initial elimination of NH3 to form an an* ion followed by elimination of Ala residues to form a series of a* ions (an*→an−1*→an−2*, etc.). This latter reaction sequence begins from a protonated C-terminal amide which has been identified by IRMPD studies as the major species present for the A-A-A-A a4 ion (Bythell et al., 2010). The present results strongly indicate that the larger an ions fragment in a manner similar to that elucidated for the all-Ala a4 ion and that the “amide” pathway is the dominant fragmentation channel for all the an ions studied in the present work.

Crystallographic analysis of NosA, which catalyzes terminal amide formation in the biosynthesis of nosiheptide.

Nosiheptide is a member of the thiopeptide family of antibiotics which demonstrates potent activities against various bacterial pathogens. The formation of its C-terminal amide is catalysed by NosA in an unusual strategy for maturating certain thiopeptides by processing precursor peptides featuring a serine extension. Here, a recombinant C-terminally truncated selenomethionine-derivatized NosA1-111 variant from Streptomyces actuosus consisting of residues 1-111, named SeMet NosA1-111, was crystallized using the sitting-drop vapour-diffusion method. Diffraction data were collected to 2.40 Å resolution using synchrotron radiation. The crystals belonged to the primitive cubic space group P4132, with unit-cell parameters a = b = c = 143.3 Å. Assuming the presence of three molecules in the asymmetric unit, the calculated Matthews coefficient was 3.94 Å(3) Da(-1) and the corresponding solvent content was 40.3%.

Hydrophobic benzyl amines as supports for liquid-phase C-terminal amidated peptide synthesis: application to the preparation of ABT-510.

C-terminal amidation is one of the most common modification of peptides and frequently found in bioactive peptides. However, the C-terminal modification must be creative, because current chemical synthetic techniques of peptides are dominated by the use of C-terminal protecting supports. Therefore, it must be carried out after the removal of such supports, complicating reaction work-up and product isolation. In this context, hydrophobic benzyl amines were successfully added to the growing toolbox of soluble tag-assisted liquid-phase peptide synthesis as supports, leading to the total synthesis of ABT-510 (2). Although an ethyl amide-forming type was used in the present work, different types of hydrophobic benzyl amines could also be simply designed and prepared through versatile reductive aminations in one step. The standard acidic treatment used in the final deprotection step for peptide synthesis gave the desired C-terminal secondary amidated peptide with no epimerization.

An Experimental and Computational Study of the Gas-Phase Acidities of the Common Amino Acid Amides.

Using proton-transfer reactions in a Fourier transform ion cyclotron resonance mass spectrometer and correlated molecular orbital theory at the G3(MP2) level, gas-phase acidities (GAs) and the associated structures for amides corresponding to the common amino acids have been determined for the first time. These values are important because amino acid amides are models for residues in peptides and proteins. For compounds whose most acidic site is the C-terminal amide nitrogen, two ions populations were observed experimentally with GAs that differ by 4-7 kcal/mol. The lower energy, more acidic structure accounts for the majority of the ions formed by electrospray ionization. G3(MP2) calculations predict that the lowest energy anionic conformer has a cis-like orientation of the [-C(═O)NH](-) group whereas the higher energy, less acidic conformer has a trans-like orientation of this group. These two distinct conformers were predicted for compounds with aliphatic, amide, basic, hydroxyl, and thioether side chains. For the most acidic amino acid amides (tyrosine, cysteine, tryptophan, histidine, aspartic acid, and glutamic acid amides) only one conformer was observed experimentally, and its experimental GA correlates with the theoretical GA related to side chain deprotonation.

The impact of model peptides on structural and dynamic properties of egg yolk lecithin liposomes - experimental and DFT studies.

Electron spin resonance (ESR), (1) H-NMR, voltage and resistance experiments were performed to explore structural and dynamic changes of Egg Yolk Lecithin (EYL) bilayer upon addition of model peptides. Two of them are phenylalanine (Phe) derivatives, Ac-Phe-NHMe (1) and Ac-Phe-NMe2 (2), and the third one, Ac-(Z)-ΔPhe-NMe2 (3), is a derivative of (Z)-α,β-dehydrophenylalanine. The ESR results revealed that all compounds reduced the fluidity of liposome's membrane, and the highest activity was observed for compound 2 with N-methylated C-terminal amide bond (Ac-Phe-NMe2 ). This compound, being the most hydrophobic, penetrates easily through biological membranes. This was also observed in voltage and resistance studies. (1) H-NMR studies provided a sound evidence on H-bond interactions between the studied diamides and lecithin polar head. The most significant changes in H-atom chemical shifts and spin-lattice relaxation times T1 were observed for compound 1. Our experimental studies were supported by theoretical calculations. Complexes EYLAc-Phe-NMe2 and EYLAc-(Z)-ΔPhe-NMe2 , stabilized by NH⋅⋅⋅O or/and CH⋅⋅⋅O H-bonds were created and optimized at M06-2X/6-31G(d) level of theory in vacuo and in H2 O environment. According to our molecular-modeling studies, the most probable lecithin site of H-bond interaction with studied diamides is the negatively charged O-atom in phosphate group which acts as H-atom acceptor. Moreover, the highest binding energy to hydrocarbon chains were observed in the case of Ac-Phe-NMe2 (2).

Photoregulation of α-Chymotrypsin Activity by Spiropyran-Based Inhibitors in Solution and Attached to an Optical Fiber.

Here the synthesis and characterization of a new class of spiropyran-based protease inhibitor is reported that can be reversibly photoswitched between an active spiropyran (SP) isomer and a less active merocyanine (MC) isomer upon irradiation with UV and visible light, respectively, both in solution and on a surface of a microstructured optical fiber (MOF). The most potent inhibitor in the series (SP-3 b) has a C-terminal phenylalanyl-based α-ketoester group and inhibits α-chymotrypsin with a Ki of 115 nM. An analogue containing a C-terminal Weinreb amide (SP-2 d) demonstrated excellent stability and photoswitching in solution and was attached to the surface of a MOF. The SP isomer of Weinreb amide 2 d is a competitive reversible inhibitor in solution and also on fiber, while the corresponding MC isomer was significantly less active in both media. The ability of this new class of spiropyran-based protease inhibitor to modulate enzyme activity on a MOF paves the way for sensing applications.

Characterization of Recombinant Monoclonal Antibody Charge Variants Using OFFGEL Fractionation, Weak Anion Exchange Chromatography, and Mass Spectrometry.

Recombinant monoclonal antibody charge heterogeneity has been commonly observed as multiple bands or peaks when analyzed by charge-based analytical methods such as isoelectric focusing electrophoresis and cation or anion exchange chromatography. Those charge variants have been separated by some of the above-mentioned methods and used for detailed characterization. The utility of a combination of OFFGEL fractionation and weak anion exchange chromatography to separate the charge variants of a recombinant monoclonal antibody was demonstrated in the current study. Charge variants were separated into various fractions of high purity and then analyzed thoroughly by liquid chromatography mass spectrometry. Analysis of intact molecular weights identified the presence of heavy chain leader sequence, C-terminal lysine, and C-terminal amidation. The identified modifications were further localized into different regions of the antibody from analysis of antibody fragments obtained from FabRICATOR digestion. Analysis of tryptic peptides from various fractions further confirmed the previously identified modifications in the basic variants. Asparagine deamidation and aspartate isomerization were identified in acidic fractions from analysis of tryptic peptides. Basic variants have been fully accounted for by the identified modifications. However, only a portion of the acidic variants can be explained by deamidation and isomerization, suggesting that additional modifications are yet to be identified or acidic variants are an ensemble of molecules with different structures.

ChemInform Abstract: Linkers, Resins, and General Procedures for Solid‐Phase Peptide Synthesis

AbstractReview: 26 refs.

Capping Motif for Peptide Helix Formation.

It is known that a C-terminal lysine stabilizes helix formation in polyalanine peptides that have seven or more residues. Using a combination of cold ion spectroscopy and DFT calculations, we demonstrate that even a three-residue peptide, Ac-Phe-Ala-LysH(+), adopts a structure in which the lysine side chain forms three hydrogen bonds with backbone carbonyls, reproducing the capping motif of larger polyalanine helices. This is confirmed by comparison with Ac-Phe-(Ala)5-LysH(+), which forms a 310 helix containing the same structural feature. In both molecules, we identified the vibrational bands of the N- and C-terminal amide NH stretches, which lack strong hydrogen bonds with carbonyls and consequently appear in a characteristic region above 3400 cm(-1). A similar pattern is also present in the even longer peptide Ac-Phe-(Ala)10-LysH(+), illustrating the generality of this capping motif. The two longer peptides contain additional, characteristic amide NH stretch bands below 3400 cm(-1), which form the core of the helix.