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.     

D ˙ Ala3 analog of elastin polypentapeptide.

The polypentapeptide, H(L˙Val₁-L˙Pro₂-D·Ala₃-L˙Val₄-Gly₅)_n Val-OMe which is the D·Ala₃ analog of the elastomeric polypentapeptide (PPP) of elastin, (L˙Val₁-L˙Pro₂-Gly₃-L˙Val₄-Gly₅)_n, has been synthesized. Its conformation is compared to that of the PPP and found to be similar with a somewhat stabilized β-turn. The D·Ala₃ analog coacervates to form a more cohesive viscoelastic material and the coacervate when cross-linked by γ-irradiation exhibits an approximate doubling of the Young's modulus of elasticity. These results are discussed in connection with other related analogs of the polypentapeptide of elastin, which are non-elastomeric, and found to be consistent with a proposed conformationally based librational entropy mechanism of elasticity.     

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.     

Unusual thionation of a cyclic hexapeptide

One carbonyl oxygen of the cyclic hexapeptide cycle(-Gly¹-Pro²-Phe³-Val⁴-Phe⁵-Phe⁶-) (A) can be selectively exchanged with sulphur using Yokoyama's reagent. Surprisingly it was not the C=O of Gly¹ but that of Phe⁵ which was substituted and cyclo(-Gly¹-Pro62-Phe³-Va1⁴-Phe⁵ψ[CS-NH]Phe⁶-) (B)was obtained. Thionation results in a conformational change of the peptide backbone although the C=O of Phe⁵ and the corresponding C=S are not involved in internal hydrogen bonds. Two isomers in slow exchange, containing a CIS Gly¹-Pro² bond in a βVIa-turn (minor) and a trans Gly-Pro bond in a βII′-turn (major), were analyzed by restrained molecular dynamics in vacuo and in DMSO as well as using time dependent distance constraints. It is impossible to fit all experimental data to a static structure of each isomer. Interpreting the conflicting NOES, local segment flexibility is found. MD simulations lead to a dynamic model for each structure with evidence of an equilibrium between a βI- and βII-turn about the Val⁴-Phe⁵ amide bond in both the cis and trans isomers. Additionally proton relaxation rates in the rotating frame (R_1p) were measured to verify the assumption of this fast βI/βII equilibrium within each isomer. Significant contributions to R_1p-rates from intramolecular motions were found for both isomers. Therefore it is possible to distinguish between at least four conformers interconverting on different time scales based on NMR data and MD refinement. This work shows that thionation is a useful modification of peptides for conformation-activity investigations.     

Specificity of 17β-oestradiol and benzo[α]pyrene oxidation by polymorphic human cytochrome P4501B1 variants substituted at residues 48, 119 and 432

1. Eight human cytochrome P4501B1 (CYP1B1) allelic variants, namely Arg⁴⁸Ala¹¹⁹Leu⁴³², Arg⁴⁸Ala¹¹⁹Val⁴³², Gly⁴⁸Ala¹¹⁹Leu⁴³², Gly⁴⁸Ala¹¹⁹Val⁴³², Arg⁴⁸Ser¹¹⁹Leu⁴³², Arg⁴⁸Ser¹¹⁹Val⁴³², Gly⁴⁸Ser¹¹⁹Leu⁴³² and Gly⁴⁸Ser¹¹⁹Val⁴³² (all with Asn⁴⁵³), were expressed in Escherichia coli together with human NADPH-P450 reductase and their catalytic specificities towards oxidation of 17β-oestradiol and benzo[α]pyrene were determined. 2. All of the CYP1B1 variants expressed in bacterial membranes showed Fe²⁺·CO versus Fe²⁺ difference spectra with wavelength maxima at 446nm and they reacted with antibodies raised against recombinant human CYP1B1 in immunoblots. The ratio of expression of the reductase to CYP1B1 in these eight preparations ranged from 0.2 to 0.5. 3. CYP1B1 Arg⁴⁸ variants tended to have higher activities for 17β-oestradiol 4-hydroxylation than Gly⁴⁸ variants, although there were no significant variations in 17β-oestradiol 2-hydroxylation activity in these eight CYP1B1 variants. Interestingly, ratios of formation of 17β-oestradiol 4-hydroxylation to 2-hydroxylation by these CYP1B1 variants were higher in all of the Val⁴³² forms than the corresponding Leu⁴³² forms. 4. In contrast, Leu⁴³² forms of CYP1B1 showed higher rates of oxidation of benzo[α]pyrene (to the 7,8-dihydoxy-7,8-dihydrodiol in the presence of epoxide hydrolase) than did the Val⁴³² forms. 5. These results suggest that polymorphic human CYP1B1 variants may cause some altered catalytic specificity with 17β-oestradiol and benzo[α]pyrene and may influence susceptibilities of individuals towards endogenous and exogenous carcinogens.     

Comparative conformational studies on cyclic hexapeptides corresponding to message sequence His-Phe-Arg-Trp of α-Melanotropin by NMR

Solution conformation of cyclo(Gly¹-His²-Phe³-Arg⁴-Trp⁵-Gly⁶) and its d -Phe analog corresponding to the message sequence [Gly-α-MSH_5-10] of α-MSH has been studied by 1D and 2D proton magnetic resonance spectroscopy in dimethyl sulfoxide (DMSO)-d₆ solution and in a DMSO-d₆/H₂O cryoprotective mixture. The NMR data for both the analogs in solution at 300 K cannot be interpreted based on a single ordered conformation, as evidenced by the broadening of only -NH resonances as well as the temperature coefficients of the amide protons. An analysis of the nuclear Overhauser effect (NOE) cross-peaks in conjunction with temperature coefficient data indicates an equilibrium of multiple conformers with a substantial population of particular conformational states at least in the d -analog. The molecular dynamics simulations without and with NOE constraints also reveal numerous low-energy conformers with two γ-turns, a γ-turn and a β-turn, two β-turns, etc. for both the analogs. The observed NMR spectra can be rationalized by a dynamic equilibrium of conformers characterized by a γ-bend at Gly⁶, two γ-bends at Phe³ and Gly⁶ and a conformer with a single β-turn and a γ-bend for the l -Phe analog. On the other hand, a conformation with two fused β-turns around the two tetrads His²-d -Phe³-Arg⁴-Trp⁵ and Trp⁵-Gly⁶-Gly¹-His² dominates the equilibrium mixture for the d -Phe analog. For the d -Phe analog, the experimentally observed average conformation is corroborated by molecular dynamics simulations as well as by studies in cryoprotective solvent.     

Peptide conformations. 33. Conformational analysis of cyclic enkephalin analogs of the type Tyr-cyclo-(-N ω -Xxx-Gly-Phe-Leu-)*

Four conformationally restricted cyclic enkephalin analogs of the type Tyr-cyclo(-Nω -Xxx-Gly-Phe-Leu-) with Xxx = l -Orn, d -Orn, l -Lys and d -Lys have been synthesized by conventional methods and the conformation of the Bocprotected, the deprotected as well as the des Tyr¹ analogs analysed by proton and nitrogen n.m.r. spectroscopy. The assignments, of the resonances were performed by two-dimensional homo- and heteronuclear correlated spectroscopy in the normal (COSY, SECSY) as well as a modified (for the detection of small couplings) version. NOE difference spectroscopy was used to distinguish the amino acid residues with aliphatic side chains. The n.m.r. parameters suggest a rather rigid conformation of the ornithine analogs with two internal NH protons whereas the lysine peptides appear to be more flexible. The structure of the Orn²-analogs can be described by a Gly³-CO HN-Leu⁵-γⁱ-turn and a hydrogen bond Orn²-CO HN -Orn². The d -lysine compound seems to include a β-turn (Gly³-CO HN-d -Lys²). The relation of the different conformational properties of the four cyclic peptides and their biological activities are briefly discussed.     

CARBON-13-N.M.R.-SPECTRA OF ANTAMANIDE,SEVERAL ANALOGUES AND THEIR ALKALI ION COMPLEXES

Natural abundance carbon-13 Fourier transform n.m.r.-spectra were obtained of the cyclic decapeptides [Phe⁴ Val⁶] antamanide (J); antamanide (II); [Tyr⁵] antamanide (III); [Ala¹] antamanide (IV) and their ion complexes (I)-Na⁺, (II)-Na⁺, (II)-Li⁺, (III)-Na⁺ and (IV)-Na⁺, Based upon literature data, systematic comparisons and model compounds, a line assignment approach was performed for the majority of the aliphatic carbons. The spectra of the [Ala Val⁶] analogues (V) (Bystrov et al., 1972) and II (measured in CD₃CN Patel, 1973a, b) and their complexes were also assigned. Characteristic chemical shift variations observed upon complex formation were calculated (free peptide/Me⁺ complex) for a number of corresponding carbons, revealing shift changes up to 2.4 p.p.m. Preliminary calculations of the angle at selected prolines for some ion complexes are included.     

Conformation-function relationships in LHRH analogs

A systematic conformational build-up procedure was performed for the LHRH molecule, pGlu¹-His²-Trp³-Ser⁴-Tyr⁵-Gly⁶-Leu⁷-Arg⁸-Pro⁹-Gly¹⁰-NH₂. The results showed a very high flexibility of the LHRH backbone, with 300 conformers being regarded as having low energy. At the same time, the conformational flexibility of LHRH differs among the fragments of the molecule. The most rigid fragments of LHRH are the Ser⁴-Tyr⁵-Gly⁶-Leu⁷ and Tyr⁵-Gly⁶-Leu⁷-Arg⁸ central tetrapeptides, the latter possessing only eight different types of low-energy backbone conformers. These eight conformer types belong to different kinds of chain reversals which are stabilized by different systems of intramolecular hydrogen bonds. Some of them resemble the β-II′ turn, which was derived as the LHRH structure from energy calculations by others. The results obtained are in good agreement with the experimental data on LHRH flexibility in solution.     

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.     

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.     

Synthesis of 17O isotope labeled Leu-enkephalin and 17O n.m.r.*

Oxygen-17 isotope was introduced into the α-carboxyl group of glycine, 1-phenylalanine, 1-leucine and 1-tyrosine by acid catalyzed exchange of ¹⁷O from H₂¹⁷O or by acid hydrolysis of respective amino acid methyl esters in H₂¹⁷O. Quantitative enrichment of glycine was achieved by acid hydrolysis of amino acetonitrile in H₂¹⁷O. For α-amino protection in amino acids t-butoxycarbonyl (Boc) group was employed for ¹⁷O labeled enkephalin synthesis. Five analogues of Leu-enkephalins (I–V) labeled with ¹⁷O at different amino acid residues were synthesized by solid phase method. ¹⁷O n.m.r. spectra were measured at 24.4 and 67.8 MHz for Leu-enkephalins ¹⁷O labeled at Gly² and Phe⁴ positions. A downfield shift was observed for ¹⁷O labeled Gly² Leu-enkephalin upon heating. This shift is indicative of the rupture of intramolecular hydrogen bonds. The preliminary results confirm the hypothesis that an intramolecular hydrogen bond exists between the carbonyl group of Gly² and NH group of Leu⁵.     

Combined use of NMR,distance geometry and MD calculations for the conformational analysis of opioid peptides of the type [D(L)-Cys2, D(L)-Cys5]enkephalin

The solution structures of a series of conformationally restricted pentapeptides with a sequence H-Tyr¹-Cys²-Gly³ Phe⁴-Cys⁵-OH cyclic (2-5) disulfide, where the cysteines possess either the D or L configuration, were examined by a combined approach including NMR measurements as well as MD calculations. It turned out that at least one low energy conformer of H-Tyr¹-Cys²-Gly³-Phe⁴-Cys⁵-OH cyclic (2-5) disulfide (DCDCE), as well as one conformer out of the group of calculated conformers for H-Tyr¹-D-Cys²-Gly³-Phe⁴-Cys⁵-OH cyclic (2-5) disulfide (DCLCE), satisfies the NMR data obtained in this study, whereas for the derivative H-Tyr^l-Cys²-Gly³-Phe⁴-Cys⁵-OH cyclic (2-5) disulfide, which contains solely L-Cys (LCLCE), there is no single structure compatible with the NMR data. © Munksgaard 1996.     

Synthesis and opioid activity of partial retro-inverso analogs of dermorphin

We studied the effect of partial retro-inverso modification of selected peptide bonds of dermorphin (H-Tyr-d -Ala-Phe-Gly-Tyr-Pro-Ser-NH₂. The modifications concern two consecutive peptide bonds (Phe³-Cly⁴-Tyr⁵, I) or a single one (Gly⁴-Tyr⁵-, II or Phe³-Gly⁴, III). All pseudoheptapeptides showed low opioid activity in the in vitro and in vivo tests. Compound III has a biological potency comparable to that of morphine but only 2–5% of original dermorphin when tested in guinea pig ileum preparation and in mice tail-flick assay after intra-cerebro or subcutaneous administration.     

METHIONINE ENKEPHALIN AND ISOSTERIC ANALOGUES

Biological activities are reported for two different types of analogues of methionine enkephalin. Cyclic analogues, bridged between the amino- and carboxy- terminals of the parent peptide, are inactive. In contrast, significant levels of activity are displayed by linear isosterically modified analogues in which the Tyr¹ -Gly² peptide bond is replaced by either -CH2NH- or -CH2 CH2-. Similar replacements of the Gly²-Gly³ peptide bond yield compounds with much reduced potency. These modifications serve as useful probes of the receptor conformation. Based on these findings, a model is proposed for interaction between enkephalin and its receptor.     

Effect of asymmetric modification on the conformation of ascidiacyclamide analogs

Abstract: Ascidiacyclamide (ASC), cyclo(‐Ile¹‐Oxz²‐d ‐Val³‐Thz⁴‐)₂ (Oxz=oxazoline and Thz=thiazole) has a C₂‐symmetric sequence, and the relationships between its conformation and symmetry have been studied. In a previous study, we performed asymmetric modifications in which an Ile residue was replaced by Gly, Leu or Phe to disturb the symmetry [Doi et al. (1999) Biopolymers 49 , 459–469]. In this study, the modifications were extended. The Ile¹ residue was replaced by Gly, Ala, aminoisobutyric acid (Aib), Val, Leu, Phe or d ‐Ile, and the d ‐Val³ residue was replaced by Val. The structures of these analogs were analyzed by X‐ray diffraction, ¹H NMR and CD techniques. X‐Ray diffraction analyses revealed that the [Ala¹], [Aib¹] and [Phe¹]ASC analogs are folded, whereas [Val¹]ASC has a square form. These structures are the first examples of folded structures for ASC analogs in the crystal state and are similar to the previously reported structures of [Gly¹] and [Phe¹]ASC in solution. The resonances of amide NH and Thz CH protons linearly shift with temperature changes; in particular, those of [Aib¹], [d ‐Ile¹] and [Val³]ASCs exhibited a large temperature dependence. DMSO titration caused nonlinear shifts of proton resonances for all analogs and largely affected [d ‐Ile¹] and [Val³]ASCs. A similar tendency was observed upon the addition of acetone to peptide solutions. Regarding peptide concentration changes, amide NH and Thz CH protons of [Gly¹]ASC showed a relatively large dependence. CD spectra of these analogs indicated approximately two patterns in MeCN solution, which were related to the crystal structures. However, all spectra showed a similar positive Cotton effect in TFE solution, except that of [Val³]ASC. In the cytotoxicity test using P388 cells, [Val¹]ASC exhibited the strongest activity, whereas the epimers of ASC ([d ‐Ile¹] and [Val³]ASCs), showed fairly moderate activities.     

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.     

Comparative conformational analysis and in vitro pharmacological evaluation of three cyclic hexapeptide NK-2 antagonists

The conformational analysis of three cyclic hexapeptides is presented. Cyclo-(-Gln⁶-Trp⁷-Phe⁸-Gly⁹-Leu¹⁰-d -Met¹¹-) (1) and cyclo-(-Gln⁶-Trp⁷-Phe⁸-Gly⁹-Leu¹⁰-Met¹¹-) (2) are NK-2 antagonists in the hamster trachea assay, whereas cyclo-(-Gln⁶-Trp⁷-Phe⁸-(R)-Gly⁹-[ANC-2]Leu¹⁰-Met¹¹-) (3), where Gly⁹[ANC-2]Leu¹⁰ represents (2S)-2-((3R)-3-amino-2-oxo-1-pyrrolidinyl)-4-methylpentanoyl, is inactive as agonist and antagonist in this assay. In DMSO, the NMR results cannot be interpreted as being consistent with a single conformation. However, the combined interpretation of results from NMR spectroscopy, restrained molecular dynamics simulations with application of proton–proton distance information from ROESY spectra, and pharmacological results leads to a reduced number of conformational domains for each peptide, which can be compared with each other and may be classified as responsible for their biological activity. Trying to match the conformational domains approximately with regular β- and γ-turns, we find a γⁿ-turn at the position of the methionine occuring in all peptides. For the active peptides 1 and 2 we arrive at an inverse γⁱ-turn at Phe⁸, and βI′- or βII-turns with Gly⁹ and Leu¹⁰ at the corner positions, these β-turns having a similar topology with respect to the linking peptide unit. Other conformational domains common to only 1 and 2 support their classification as responsible for the biological activity.     

Synthesis and AT2 receptor‐binding properties of angiotensin II analogues

Abstract: The present study investigates the importance of the amino acid side chains in the octapeptide angiotensin II (Ang II) for binding to the AT₂ receptor. A Gly scan was performed where each amino acid in Ang II was substituted one‐by‐one with glycine. The resulting set of peptides was tested for affinity to the AT₂ receptor (porcine myometrial membranes). For a comparison, the peptides were also tested for affinity to the AT₁ receptor (rat liver membranes). Only the substitution of Arg² reduced affinity to the AT₂ receptor considerably (92‐fold when compared with Ang II). For the other Gly‐substituted analogues the affinity to the AT₂ receptor was only moderately affected. To further investigate the role of the Arg² side chain for receptor binding, we synthesized some N‐terminally modified Ang II analogues. According to these studies a positive charge in the N‐terminal end of angiotensin III [Ang II (2–8)] is not required for high AT₂ receptor affinity but seems to be more important in Ang II. With respect to the AT₁ receptor, [Gly²]Ang II and [Gly⁸]Ang II lacked binding affinity (K_i > 10 μm ). Replacement of the Val³ or Ile⁵ residues with Gly produced only a slight decrease in affinity. Interestingly, substitution of Tyr⁴ or His⁶, which are known to be very important for AT₁ receptor binding, resulted in only 48 and 14 times reduction in affinity, respectively.