d· Ala5 analog of the elastin polypentapeptide. Physical characterization

The d ° Ala₅ analog, (l · Val₁-l · Pro₂-Gly3-l · Val₄-d · Ala₅)_n, of the polypentapeptide (PPP) of elastin is synthesized and characterized by a series of physical methods. Carbon-13 and proton nuclear magnetic resonance spectroscopies are used to verify purity and, by means of solvent dependence of peptide C -O chemical shift and of temperature dependence of peptide NH chemical shift, to establish by comparison with the PPP of elastin the presence and increased stability of the Type II Pro₂-Gly₃ β-turn. The temperature dependence of aggregation in water to form a viscoelastic phase called the coacervate is reported for several concentrations. Comparison of carbon-13 nuclear magnetic resonance spectra obtained under identical conditions for the coacervate states of the PPP of elastin and the d · Ala₅ analog shows the effect of replacing the Gly₅ residue by a d · Ala_s residue to be one of greatly restricting mobility of the polypeptide chain. Scanning electron micrographs, of the coacervate alone and of the coacervate cross-linked and compounded to a Dacron fabric before and after stress-strain studies, are reported which show the d · Ala₅ PPP matrix to rupture during the stresses of drying and of stretching while wet. Thus, the effect of adding a methyl moiety to the Gly₅ residue of the PPP of elastin is to decrease markedly the mobility of the polypeptide chain and to destroy elasticity. The results are presented as a test of the proposed librational entropy mechanism of elasticity of the PPP of elastin.     

CONFORMATIONAL CHARACTERIZATION OF CYCLOPENTAPEPTIDE,LVAL-LPRO-GLY-LVAL-GLY: A REPEATING ANALOGUE OF ELASTIN

The cyclopentapeptide, ·L·Val¹-L· Pro²-Gly³-L· Val⁴-Gly⁵, was synthesized and its conformational characterization was carried out using n.m.r. and theoretical energy calculations. The n.m.r. studies indicated the existence of a cis Val¹-Pro² peptide bond in water and a very strong intramolecular H-bond between the Val¹ NH and Gly³ C=O groups. This H-bond forms a β-turn (type II) placing Val⁴ Gly⁵ residues within the turn. Two minimum energy conformations were derived, one of which agrees very well with the solution conformation.     

STUDIES ON THE CONFORMATION AND INTERACTIONS OF ELASTIN: NUCLEAR MAGNETIC RESONANCE OF THE POLYHEXAPEPTIDE

Synthesis, proton magnetic resonance and carbon-13 magnetic resonance characterizations, including complete assignments, are reported for the polyhexapeptide of elastin, HCO-Val-(Ala₁-Pro₂-Gly₃-Val₄-Gly₅-Val₆)₁₈-OMe. Temperature dependence of peptide NH chemical shifts and solvent dependence of peptide C-O chemical shifts have been determined in several solvents and have been interpreted in terms of four hydrogen bonded rings for each repeat of the polyhexapeptide. The more stable hydrogen bonded ring is a β-turn involving Ala₁ C-O…HN·Val₄ More dynamic hydrogen bonds are an 11-atom hydrogen bonded ring Gly₃ NH · O-C Gly₅, a 7-atom hydrogen bonded ring (a γ-turn) Gly₃ C-O … HN · Gly₅ and a 23-atom hydrogen bonded ring Val₆₁NH … O-C Val_6(l+l). This set of hydrogen bonds results in a right-handed β-spiral structure with slightly more than two repeats (approximately 2.2) per turn of spiral. The β-spiral structure is briefly discussed relative to data on the elastic fiber.     

NON-ELASTOMERIC POLYPEPTIDE MODELS OF ELASTIN

The synthesis of two polyhexapeptides is reported. The polyhexapeptides are H-(Val⁶-Ala¹-Pro²-Gly³-θ⁴-Gly⁵)_n -Val-OMe where θ= Vol or Lys with a mole ratio of 3.5:1 and H-(Val⁶-Ala¹-Pro²-Gly³-Lys⁴-Gly⁵)_n-Val-OMe. The first polymer was utilized with a previously synthesized polyhexapeptide, H-(Val⁶-Ala¹ -Pro²-Gly³-θ-Gly⁵)_n-Val-OMe where θ was either Val⁴ or Glu⁴ at a mole ratio of 3.5:1, to obtain an intermolecular cross-linked matrix by means of primary amide bond formation between the γ-carboxyls of the Glu residues of one copolymer and the α-amino groups of the Lys residues of the other copolymer. The cross-linking reaction was run during a temperature-elicited phase separation with flow orientation of the polymers. An insoluble, non-elastomeric, cross-linked, polyhexapeptide matrix was obtained. The nature of the insoluble polyhexapeptide matrix was well-demonstrated by the polymer, H-(Val⁶-Ala¹-Pro²-Gly³-θ⁴-Gly⁵)_n-Val-OMe where θ⁴ is Vol or Lys, which could be formed into cellophane-like, non-elastomeric sheets which would tear and which could be shown by microscopy to have sharp edges. This very different property of the polyhexapeptide of tropoelastin as compared to the elastomeric polypentapeptide of tropoelastin is discussed in terms of a different structural role. The purity of key intermediate hexamers and of the polyhexapeptides is demonstrated by carbon-13 magnetic resonance in addition to the usual analytical methods.     

Polytetrapeptide of elastin

High molecular weight polytetrapeptide of elastin, (L-Val¹-L-Pro²-Gly³-Gly⁴)_n, was synthesized using activation of the (GGVP) permutation for polymerization. The temperature-dependence of aggregation was characterized as a function of concentration and the circular dichroism spectra were obtained in the 20° to 70°C temperature range. The latter showed an inverse temperature transition centered near 50°C in which polypeptide order increased on raising the temperature. A concentration of 0.6 g of polytetrapeptide in 1 g of water was Λ irradiation cross-linked (20 Mrad) to form an elastomeric matrix. A study of the temperature-dependence of elastomeric force demonstrated a transition toward increased force on raising the temperature with a midpoint of the transition near 50°C. Thus, there is a correlation between increase in intramolecular order and elastomeric force development. These results are compared to previous results on the polypentapeptide of elastin, (VPGVG)_n and on an analog, (IPGVG)_n, to demonstrate that the temperature of the transition is proportional to the hydrophobicity of the repeating unit. The point is noted that the elastomeric force development correlates better with intramolecular ordering than with intermolecular processes.     

β-ENDORPHIN: SYNTHESIS AND ANALGESIC ACTIVITY OF SEVERAL ANALOGS MODIFIED IN POSITIONS 2 AND 5

The solid-phase syntheses of [Sar²]-, [Ala²]-, [D-Leu²]-, [D-Lys²]-β- endorphins and [Pro⁵]-, [Leu⁵]-, [D-Leu⁵]-, [D-Ala², D-Leu⁵]-β-endorphins are described. The synthetic peptides were purified by chromatography on carboxymethylcellulose and partition chromatography on Sephadex G-50. They were characterized by partition chromatography on agarose, thin-layer chromatography, paper electrophoresis, and amino acid analyses of acid and enzymic hydrolysates. Bioassay of the synthetic analogs for analgesic activity by the tail-flick method showed the D-Leu² analog to be 48% as potent as β_h-endorphin while the Ala², D-Lys², Leu⁵, and [D-Ala², D-Leu⁵] analogs were 8 to 17% as active. The Sar², D-Leu⁵, and Pro⁵ analogs were less than 1% as potent.     

Stabilization of β-turn conformations in enkephalins

Stereochemical constraints have been introduced into the enkephalin backbone by substituting α-aminoisobutyryl (Aib) residues at positions 2 and 3, instead of Gly. ¹H n.m.r. studies of Tyr-Aib-Gly-Phe-Met-NH₂, Tyr-Aib-Aib-Phe-Met-NH₂ and Tyr-Gly-Aib-Phe-Met-NH₂ demonstrate the occurrence of folded, intramolecularly hydrogen bonded structures in organic solvents. Similar conformations are also favoured in the corresponding t-butyloxycarbonyl protected tetrapeptides, which lack the Tyr residue. A β-turn centred at positions 2 and 3 is proposed for the Aib²-Gly³analog. In the Gly²-Aib³analog, the β-turn has Aib³-Phe⁴as the corner residues. The Aib²-Aib³analog adopts a consecutive β-turn or 3₁₀ helical conformation. High in vivo biological activity is observed for the Aib²and Aib²-Aib³analogs, while the Aib³peptide is significantly less active.     

Importance of the leucine side-chain to the spasmogenic activity and binding of Substance P analogues

Previous studies with Substance P (SP) antagonists (GR 71251, [d Pro⁹, Pro¹⁰, Trp¹¹]SP and d Pro⁹, MeLeu¹⁰, Trp¹¹]SP) have suggested the existence in the guinea-pig ileum (GPI) of two distinct tachykinin receptors associated with the contractile responses of [Pro⁹]SP and septide. In addition [Apa^9-10]SP, a glycine-substituted analogue of SP with a carba bond between residues 9 and 10, [Gly⁹-ψ(CH₂-CH₂)-Gly¹⁰SP = [Apa^9-10]SP, was shown to belong to the ‘septide family’ (low affinity for NK-1 specific binding sites and high potency in the GPI). In order to establish the importance of the isopropyl side-chain in position 10, the binding potencies and activities of [Gly⁹-ψ(CH₂-CH₂)-Gly¹⁰]SP, [Ala¹⁰]SP, [Gly⁹-ψ(CH₂-CH₂)-Leu¹⁰]SP and [Gly⁹-ψ(CH₂-CH₂)-d Leu¹⁰]SP were compared. Conformational behaviour of active peptides with a carba bond was analyzed by NMR and modelisation studies. This study with agonists demonstrated that undecapeptides substituted in position 10 in the SP sequence also enabled discrimination of NK-1 receptors from receptors responsible for the spasmogenic activities of peptides belonging to the ‘septide family’. [Gly⁹-ψ(CH₂-CH₂)- Leu¹⁰]SP is ahighly potent NK-1 agonist, [Gly⁹-ψ(CH₂-CH₂)-Gly¹⁰]SP acts on the septide-sensitive receptor, and [Ala¹⁰]SP is a mixed agonist.     

Turn induction by N-aminoproline Comparison of the Gly-Pro-Gly and Glyψ[CO-NH-N]Pro-Gly sequences

The folded structure induced by the N-aminoproline residue (the hydrazino analogue of proline, denoted hPro) in the Boc-Gly¹-hPro²-Gly³-NHiPr hydrazino tripeptide has been characterized in the solid state by X-ray diffraction, and compared to the usual βII-turn structure in the Boc-Gly¹-Pro²-Gly³⁶-NHiPr cognate tripeptide. It is stabilized by a bifurcated hydrogen bond in which (Gly³)NH interacts with both (Gly¹)CO and (hPro²)N^x. This conformation is retained in CH₂Cl₂ and CHC1₃ solutions, and allows an overall folded conformation of the hydrazino tripeptide in which (iPr)NH is hydrogen-bonded to (Boc)CO. The hPro α-hydrazino acid residue appears to promote a local folded structure, and might behave as a β-turn mimic. © Munksgaard 1994.     

CD-n.m.r. study of the solution conformation of bradykinin analogs containing α-aminoisobutyric acid

The conformation in aqueous solution of several α-aminoisobutyric acid (AIB)-containing analogs of bradykinin (BK) has been probed by complementary CD and ¹H n.m.r. measurements. The conclusion reached is that substitution of AIB for Pro² and/or Pro³ in BK stabilizes a degree of β-turn conformation in the N-terminal tetrapeptide moiety of the resulting analogs. Changing the solvent from water to DMSO or TFE further enhances the contribution of particular hydrogen bonded structures to the time-averaged conformation of these peptides. Bradykinin and [AIB⁷]-BK adopt similar hydrogen bonded conformations in TFE, apparently with a contribution from a β-turn involving their common Arg¹-Pro²-Pro³-Gly⁴ moiety. The contrasting biological activities of BK and its AIB-analogs are considered in terms of the conformational analogy between the AIB-residue and cis¹ Pro and the propensity for a β-turn at the N-terminus of the peptide.     

Identification of five pyrrolidinyl substituted cathinones and the collision‐induced dissociation of electrospray‐generated pyrrolidinyl substituted cathinones

This article reports on the analytical properties of five pyrrolidinyl substituted cathinones: α ‐pyrrolidinononaphenone (α ‐PNP, 1 ), 4‐chloro‐α ‐pyrrolidinopropiophenone (4‐Cl‐α ‐PPP, 2 ), 4‐chloro‐α ‐pyrrolidinovalerophenone (4‐Cl‐α ‐PVP, 3 ), 5‐dihydrobenzofuranpyrovalerone (5‐DBFPV, 4 ), and 2‐(pyrrolidin‐1‐yl)‐1‐(5,6,7,8‐tetrahydronaphthalen‐2‐yl)hexan‐1‐one (β ‐THNPH, 5 ). These identifications were based on liquid chromatography–quadrupole time‐of‐flight‐mass spectrometry (LC–QTOF–MS), gas chromatography–mass spectrometry (GC–MS) and nuclear magnetic resonance spectroscopy (NMR). To our knowledge, no analytical data about α ‐PNP, 4‐Cl‐α ‐PPP, 4‐Cl‐α ‐PVP, and β ‐THNPH have appeared until now, making this the first report on these compounds. Moreover, in order to study the collision‐induced dissociation (CID) characteristic fragmentation routes of pyrrolidinyl substituted cathinones, a total number of 13 pyrrolidinyl substituted cathinones were selected and discussed. The major fragmentation pathways under CID mode are produced, leading to the formation of characteristic ions. Product ions of [M‐C₄H₉N]⁺ and C_nH_2nN⁺ indicate the presence of pyrrolidinyl substitution. Characteristic fragments are also produced via the cleavages of the CH–N(CH₂)₄ bond and the CO‐CHN bond. Copyright © 2016 John Wiley & Sons, Ltd.     

[Ala12]MCD peptide: a lead peptide to inhibitors of immunoglobulin E binding to mast cell receptors1

Abstract: An effort was made to discover mast cell degranulating (MCD) peptide analogs that bind with high affinity to mast cell receptors without triggering secretion of histamine or other mediators of the allergic reaction initiated by immunoglobulin E (IgE) after mast cell activation. Such compounds could serve as inhibitors of IgE binding to mast cell receptors. An alanine scan of MCD peptide reported previously showed that the analog [Ala¹²]MCD was 120‐fold less potent in histamine‐releasing activity and fivefold more potent in binding affinity to mast cell receptors than the parent MCD peptide. Because this analog showed marginal intrinsic activity and good binding affinity it was subsequently tested in the present study as an IgE inhibitor. In contrast to MCD peptide, [Ala¹²]MCD showed a 50% inhibition of IgE binding to the FcεRIα mast cell receptor by using rat basophilic leukemia (RBL‐2H3) mast cells and fluorescence polarization. Furthermore, in a β‐hexosaminidase secretory assay, the peptide also showed a 50% inhibition of the secretion of this enzyme caused by IgE. An attempt was made to relate structural changes and biologic differences between the [Ala¹²]MCD analog and the parent MCD peptide. The present results show that [Ala¹²]MCD may provide a base for designing agents to prevent IgE/FcεRIα interactions and, consequently, allergic conditions.     

Preparation and properties of novel gramicidin S analogs possessing a tri-, tetra- or pentamethylene bridge between ornithine side chains

Abstract: Gramicidin S (GS) analogs in which the N^δ atoms of the two Orn side chains are linked by an oligomethylene bridge [-(CH₂)_n-; n=3–5] were prepared via the bis(p-nitrobenzenesulfonyl) derivative [Orn(NBS)^2,2′]GS. For comparison the nonbridged secondary amino group-containing analog [Orn(Me)^2,2′]GS was also prepared. ¹H NMR and CD spectral analysis indicated that these analogs adopt the same β-sheet conformation as GS. The antimicrobial activities of these analogs were very similar, but were slightly dependent on the bridge chain length, the trimethylene-bridged analog being the most potent.     

Inhibition of glycogen synthase kinase 3β prevents peroxide‐induced collapse of mitochondrial membrane potential in rat ventricular myocytes

1. Preconditioning has been proposed to protect the myocardium by inhibiting glycogen‐synthase kinase (GSK) 3β. The aim of the present study was to test whether transfection of ventricular myocytes with inactive GSK3β would mimic preconditioning and whether a constitutively active form of GSK3β would prevent protection by an opioid receptor agonist. 2. Isolated ventricular myocytes from adult rats were infected with live adenovirus containing either a wild‐type (wtGSK), constitutively active (caGSK) or dominant‐negative (dnGSK) GSK3β plasmid. Cells were loaded with tetramethylrhodamine ethyl ester (TMRE) and exposed to H₂O₂ (100 μmol/L) for 40 min before mitochondrial membrane potential (ΔΨ_m) was assessed using flow cytometric analysis. 3. Fluorescence intensity was reduced in H₂O₂‐treated cells compared with untreated cells, presumably because oxidant injury opened mitochondrial permeability transition pores, causing mitochondria to lose TMRE. The selective GSK3β inhibitor SB216763, as well as the δ‐opioid receptor agonist [d ‐Ala²‐d ‐Leu⁵]‐enkephalin (DADLE) (1 μmol/L), protected cells against peroxide‐induced loss of ΔΨ_m. 4. Cells transfected with dnGSK (1 μmol/L) were equally protected against peroxide stress, when given throughout the TMRE and H₂O₂ treatment, confirming a protective effect of GSK3β with a highly selective inhibition. Cells transfected with wtGSK did not show any difference in responses to H₂O₂, SB216763 or DADLE compared with untransfected cells, suggesting that adenovirus infection itself had no effect. In contrast, caGSK‐transfected myocytes could no longer be protected with DADLE, suggesting a role for GSK3β between the surface receptor and the mitochondria. 5. These experiments confirm that inhibition of GSK3β protects the myocytes, but also that the preconditioning mimetic DADLE loses its protective effect when a constitutively active GSK3β is present.     

The crystal structure of Boc-Pro-Val-Gly-NH2, with a remark on the conformation of the valyl residues in peptides

The crystal structure of Boc-Pro-Val-Gly-NH₂ has been determined: monoclinic; P2₁; a = 9.331 (3) Å, b = 9.532 (4), c = 23.080 (9), β= 91.33 (3)R, Z = 4; R = 0.053 for 3400 reflections with ˙Fo˙,>α(Fo). There are two independent but very similar molecules in the crystal. The peptide main chains are in an extended form, and packed in two kinds of antiparallel β sheets, the (φ, Φ) angles of the central Val residues are (-156°, 146°) and (-139°, 155°), and the mean length of the N- H . 0 hydrogen bonds in the sheets is 2.965 Å. A detailed study of the conformations of the Val residues in oligopeptide crystals shows that the preferred conformation of Val in peptides is: the (φ, Φ) angles close to those of the antiparallel β sheet, and C^γ1 and C^γ2, against N with respect to the C^α– C^β bond, at either (trans, gauche) or (-gauche, gauche). The mean π(NC^αC') angle of such Val residues is 107.9(9)°. A twisting in the β sheets is also discussed.     

The synthetic cathinone psychostimulant α‐PPP antagonizes serotonin 5‐HT2A receptors: In vitro and in vivo evidence

Synthetic cathinones (SCs) are β‐keto analogs of amphetamines. Like amphetamines, SCs target monoamine transporters; however, unusual neuropsychiatric symptoms have been associated with abuse of some SCs, suggesting SCs might possess additional pharmacological properties. We performed radioligand competition binding assays to assess the affinities of nine SCs at human 5‐HT_2A receptors (5‐HT_2AR) and muscarinic M₁ receptors (M₁R) transiently expressed in HEK293 cells. None of the SCs exhibited affinity at M₁R (minimal displacement of [~K_d] [³H]scopolamine up to 10 μM). However, two SCs, α‐pyrrolidinopropiophenone (α‐PPP) and 4‐methyl‐α‐PPP, had low μM K_i values at 5‐HT_2AR. In 5‐HT_2AR–phosphoinositide hydrolysis assays, α‐PPP and 4‐methyl‐α‐PPP displayed inverse agonist activity. We further assessed the 5‐HT_2AR functional activity of α‐PPP, and observed it competitively antagonized 5‐HT_2AR signaling stimulated by the 5‐HT₂R agonist (±)‐2,5‐dimethoxy‐4‐iodoamphetamine (DOI; K_b = 851 nM). To assess in vivo 5‐HT_2AR activity, we examined the effects of α‐PPP on the DOI‐elicited head‐twitch response (HTR) in mice. α‐PPP dose‐dependently blocked the HTR with maximal suppression at 10 mg/kg (P < 0.0001), which is a moderate dose used in studies investigating psychostimulant properties of α‐PPP. To corroborate a 5‐HT_2AR mechanism, we also tested 3,4‐methylenedioxy‐α‐PPP (MDPPP) and 3‐bromomethcathinone (3‐BMC), SCs that we observed had 5‐HT_2AR K_is > 10 μM. Neither MDPPP nor 3‐BMC, at 10 mg/kg doses, attenuated the DOI HTR. Our results suggest α‐PPP has antagonist interactions at 5‐HT_2AR in vitro that may translate at physiologically‐relevant doses in vivo. Considering 5‐HT_2AR antagonism has been shown to mitigate effects of psychostimulants, this property may contribute to α‐PPPs unpopularity compared to other monoamine transporter inhibitors.     

Effect of the environment and role of the π-π stacking interactions in the stabilization of the 310-helix conformation in dehydroalanine oligopeptides

A quantum-mechanical study of the chain-length dependent stability of the extended, 2₇ribbon and 3₁₀-helix conformations in dehydroalanine (ΔAla) oligopeptides has been performed. To address the study, the oligopeptides ΔAla_n, where n varies from 1 to 6, were computed by using the semiempirical AM1 methodology. Cooperative free-energy effects permit one to predict the stabilization of the 3₁₀-helix with respect to the extended and 2₇-ribbon conformations when the number of residues in the polypeptide chain increases. The interactions associated with the π-electron density of the side chains can easily explain this finding. The effects of the solvent and the crystalline packing on the different conformations were modeled using a self-consistent reaction field (SCRF) method and a molecular mechanics approach to the packing, respectively. Both the aqueous and crystal environments seem to be a key factor in the stabilization of the helical conformation. Finally, the variations of electrostatic parameters such as atomic point charges and dipole moments in ΔAla-containing peptides with internal (conformation) and external (solvent) effects are discussed. © Munksgaard 1995.     

[1-Desaminopenicillamine, 8-α-hydroxyisocaproic acid] oxytocin. A selective inhibitor in rats of the uterine response to oxytocin

[1-Desaminopenicillamine, 8-α-hydroxyisocaproic acid] oxytocin was synthesized by a 6 + 3 fragment condensation from precursors which had been formed by solution methods. This analog inhibited uterine responses to oxytocin (pA₂ 7.37, 7.9, 6.17; uterus in vitro without Mg++, in vitro with Mg++, and in vivo, respectively) and showed little or no activity in other bioassays.     

The 10th and 11th residues of short length N‐terminal PTH(1–12) analogue are important for its optimum potency

Abstract: The 10th and 11th residues of parathyroid hormone PTH(1–12) analogues were substituted to study the structure and function of PTH analogues. The substitution of Ala¹⁰ of [Ala^3,10,12(Leu⁷/Phe⁷)Arg¹¹]rPTH(1–12)NH₂ with Glu¹⁰ and/or the Arg¹¹ with Ile¹¹ markedly decreased cAMP generating activity. Data from circular dichroism (CD) and the nuclear magnetic resonance (NMR) structural analysis of [Ala^3,10,12(Leu⁷/Phe⁷)Arg¹¹]rPTH(1–12)NH₂ revealed tight α‐helical structures, while the Glu¹⁰ and/or Ile¹¹ substituted analogues showed unstable α‐helical structures. We conclude that 10th and 11th residues are important for stabilizing its helical conformation and that destabilization of the α‐helical structure, induced by substituting the above residues, remarkably affect its biological potency.     

Conformational analysis of endomorphin‐1 by molecular dynamics methods

Abstract: Endomorphin‐1 (EM1, H‐Tyr‐Pro‐Trp‐Phe‐NH₂) is a highly potent and selective agonist for the μ‐opioid receptor. A conformational analysis of this tetrapeptide was carried out by simulated annealing and molecular dynamics methods. EM1 was modeled in the neutral (NH₂‐) and cationic (NH‐) forms of the N‐terminal amino group. The results of NMR measurements were utilized to perform simulations with restrained cis and trans Tyr¹‐Pro² peptide bonds. Preferred conformational regions in the Φ₂–Ψ₂, Φ₃–Ψ₃ and Φ₄–Ψ₄ Ramachandran plots were identified. The g(+), g(?) and trans rotamer populations of the side‐chains of the Tyr¹, Trp³ and Phe⁴ residues were determined in χ₁ space. The distances between the N‐terminal N atom and the other backbone N and O atoms, and the distances between the centers of the aromatic side‐chain rings and the Pro² ring were measured. The preferred secondary structures were determined as different types of β‐turns and γ‐turns. In the conformers of trans‐EM1, an inverse γ‐turn can be formed in the N‐terminal region, but in the conformers of cis‐EM1 the N‐terminal inverse γ‐turn is absent. Regular and inverse γ‐turns were observed in the C‐terminal region in both isomers. These β‐ and γ‐turns were stabilized by intramolecular H‐bonds and bifurcated H‐bonds.