1H-NMR studies of sarmesin and [des1]sarmesin conformation in dimethylsulfoxide by nuclear Overhauser effect (NOE) enhancement spectroscopy: folding of the N- and C-terminal domains

The conformational properties of the competitive angiotensin II antagonist sarmesin [Sar-Arg-Val-Tyr(Me)-His-Pro-Phe] and its heptapeptide analogue [des1]sarmesin in dimethylsulphoxide-d6 were investigated by nuclear Overhauser effect (NOE) enhancement studies. Assignment of all backbone and side-chain protons was possible by combining information from intraresidue NOE studies with two-dimensional correlated spectroscopy (COSY) studies. Saturation of the His C alpha proton of sarmesin produced essentially the same interresidue NOE enhancement of the two Pro C delta protons, illustrating the presence of the trans His-Pro bond. Saturation of the Sar N-methyl group caused enhancement of one of the His C beta protons, suggesting the presence of a turn in the N-terminal region of the molecule. Saturation of His C2 in sarmesin and [des1]sarmesin enhanced the Tyr(Me) methyl signal. Saturation of the Tyr(Me) methyl protons in [des1]sarmesin produced NOE enhancement of the His C2 and C4 protons, and saturation of the His C2 proton enhanced the Tyr(Me) meta and ortho proton signals. Interresidue interactions between the Tyr(Me) and His protons in sarmesin and [des1]sarmesin illustrate that these two side-chains remain in close proximity even in the absence of the postulated hydrogen bond between Tyr hydroxyl and the His imidazole ring in angiotensin II. The data suggest a preferred conformation for sarmesin in DMSO in which the peptide backbone is S-shaped and similar to that for angiotensin II.     

Study of bradykinin conformation in a dimethylsulfoxide solution by two-dimensional 1H-NMR spectroscopy

The role of charged groups of the nonapeptide bradykinin in stabilization of its spatial structure in dimethyl sulfoxide solution was investigated. The signal assignment in the 1H-NMR spectra was achieved by means of two dimensional correlated spectroscopy (COSY) and nuclear Overhauser enhancement spectroscopy (NOESY). The changes in the NH and C alpha H proton chemical shifts of the Arg1 and Arg9 residues, variations both in temperature coefficients of chemical shifts of NH-resonances and coupling constants, as well as the appearance of additional NOE cross-peaks in NOESY spectra for d alpha N and d beta N 1H-1H distances were revealed by comparing the NMR spectra for two states--with the protonated C-terminal carboxyl group and deprotonated one. The experimental results are in agreement with the assumption that the conformation of the peptide in (CD3)2SO is stabilized by electrostatic interaction between the oppositely charged N- and C-terminal groups. The conformation with deprotonated alpha-carboxyl group is characterized by two beta-turns in the sequences Pro2-Pro-Gly-Phe5 and Ser6-Pro-Phe-Arg9.     

Mutagenesis of the AT1 receptor reveals different binding modes of angiotensin II and [Sar1]-angiotensin II

Homology modeling of the structure of the AT1 receptor, based on the high resolution rhodopsin crystal structure, indicated that it is unlikely that the binding of AngII to AT1 involves simultaneously all the receptor's residues reported in the literature to participate in this process. Site-directed mutagenesis using Ala substitution of charged residues Lys20, Arg23, Glu91 and Arg93 was performed to evaluate the participation of their side-chains in ligand binding and in triggering the cell's response. A comparative analysis by competition binding and functional assays using angiotensin II and the analog [Sar1]-angiotensin II suggests an important role for Arg23 of AT1 receptor in binding of the natural agonist. It is discussed whether some receptor's residues participate directly in the binding with AngII or whether they are part of a regulatory site.     

Conformational analysis of [Cpp1, Sar7, Arg8] vasopressin by 1H-NMR spectroscopy and molecular mechanics calculations.

A combined 1H-NMR and molecular mechanics study of [Cpp1, Sar7]AVP was performed in order to select the most probable conformations in DMSO solutions. The NMR constraints obtained were employed in the selection of starting conformations of the cyclic moiety of the analog. In particular, the diminished accessibility of the Asn5 NH proton to solvent and the close contact between Cpp1 and Cys6 C alpha H protons suggests a beta-turn conformation at the Phe3-Gln4 residues. Energy minimization was carried out both in the ECEPP/2 (rigid-valence geometry) and in the AMBER (flexible-valence geometry) force fields. Comparison of the experimental and calculated values of NMR characteristics has revealed that conformations containing type I, II, and III beta-turns at the Phe3-Gln4 residues are in reasonable agreement with the experimental data, with a dynamic equilibrium between the beta I (beta III) and beta II type structures of the cyclic part being the most probable. All of these conformations prefer the negative chirality of the disulfide bridge (theta 3 approximately -90 degrees). Five representative conformations were chosen for the acyclic tail: one with a beta I, one with a beta II'-turn at the Sar7-Arg8 residues, two extended-type conformations, and a conformation with a gamma-turn at Sar7. Because only high-energy extended conformations were in agreement with NMR data, it was concluded that the acyclic tail has considerable conformational flexibility in solution. The conformations obtained are discussed in terms of the structure-function relationship of the neurohypophyseal hormone analogs.     

Comparative studies of angiotensin II and its analog [Sar1Ala8]angiotensin II on the density of cellular monolayer of cell cultures from green monkey kidney

The effects of the octapeptide angiotensin II (AT II) and its analog [Sar1Ala8]AT II on the cell density in cell culture from green monkey kidney (GMK) were studied. AT II and [Sar1Ala8]AT II provoked a decrease of the number of living cells depending on the concentration (0.001, 0.01 and 0.1 nM) and time of incubation (24, 48 and 72 hours), both peptides having a very similar activity. These data indicate that AT II and [Sar1Ala8]AT II may act on the same class of angiotensin receptors in GMK cells.     

Conformational analysis of r(CGCGCG) in aqueous solution: an A-type double helical conformation studied by two-dimensional nuclear Overhauser effect spectroscopy.

The conformation of the hexanucleoside pentaphosphate r( CGCGCG ) in aqueous solution was studied by circular dichroism, 1H- and 31P-NMR spectroscopy. The base-, H1'- and H2'-proton resonances were assigned by means of 2D-NOE spectroscopy. The base- and H1'-proton chemical shifts were studied as a function of temperature. Proton-proton distances are computed in A- and A'-RNA as well as in A-, B- and Z-DNA. A qualitative interpretation of the observed 2D-NOE intensities shows that r( CGCGCG ) adopts a regular A-type double helical conformation under our experimental conditions. The CD- and 31P-NMR experiments described in this paper are in agreement with this structure both under low- and high-salt conditions.     

Biological activity of the novel cyclic angiotensin II analogue [Sar1,Lys3,Glu5]ANG II

Summary This study was undertaken to investigate the biological activity of the cyclic amide-linked analogue of angiotensin II (ANG II), [Sar¹,Lys³,Glu⁵]ANG II, in both ex vivo and in vivo experiments. This constrained analogue was designed on the basis of a recently suggested conformational model for ANG II-induced receptor activation, which is characterized by a Tyr-Ile-His backbone bend and the clustering of the three aromatic rings (Tyr, His, Phe). After [Sar¹,Lys³,Glu⁵]ANG II was found to have contractile activity (15% of ANG II in the rat uterus assay), it was administered in anesthetized rabbits where it produced an immediate and dose-dependent increase in blood pressure, which peaked within minutes, was sustained as long as the drug was given, and was gradually returned to baseline after discontinuation of the drip. The blood pressure response to the cyclic analogue was of less magnitude compared to that elicited by an isovolemic and equimolar solution of ANG II. These data confirm the importance of a properly oriented ring cluster, allowing the charge-relay conformation proposed for ANG II.     

Conformational analysis of a segment in bacterioopsin by two-dimensional (1)H-NMR spectroscopy]

The spatial structure of a synthetic peptide, an analogue of the membrane spanning segment B (residues 34-65) of bacterioopsin from Halobacterium halobium, has been refined. Backbone torsion angles were derived from intensities of short-range interproton NOEs. These, together with a complete set of the NOEs integral intensities formed the basis for the three-dimensional structure refinement by the energy minimization with consideration of NOE penalty functions. Analysis indicates the right-handed alpha-helical conformation of segment B extending from Asp-38 to Tyr-64 with a kink of the helical axis (27 degrees) at Pro-50. The most stable region with an average root-mean-square deviation of 0.43 A between the backbone atoms includes residues 42-60 in six energy refined structures. The N-terminal part of segment B (residues 34-37) has no ordered conformation. The inferred structure is in close agreement with the electron cryomicroscopy structure of bacteriorhodopsin, differing from it in conformations of most of the side chains.     

1H two-dimensional nuclear Overhauser effect and relaxation studies of poly(dA).poly(dT)

The structure of poly(dA).poly(dT) in aqueous solution has been studied by using 1H two-dimensional nuclear Overhauser effect (2D NOE) spectroscopy and relaxation rate measurements on the imino and nonexchangeable protons. The assignments of the 1H resonances are determined from the observed cross-relaxation patterns in the 2D NOE experiments. The cross-peak intensities together with the measured relaxation rates show that the purine and pyrimidine strands in poly(dA).poly(dT) are equivalent in aqueous solution. The results are consistent with a right-handed B-form helix where the sugars on both strands are in the C2'-endo/anti configuration. These observations are inconsistent with a proposed heteronomous structure for poly(dA).poly(dT) [Arnott, S., Chandrasekaran, R., Hall, I. H., & Puigjaner, L. C. (1983) Nucleic Acids Res. 11, 4141-4155]. The measured relaxation rates also show that poly(dA).poly(dT) has fast, large-amplitude local internal motions (+/- 20-25 degrees) in solution and that the amplitudes of the base and sugar motions are similar. The motion of the bases in poly(dA).poly(dT) is also similar to that previously reported for poly(dA-dT).poly(dA-dT) and poly(dG-dC).poly(dG-dC) [Assa-Munt, N., Granot, J., Behling, R. W., & Kearns, D. R. (1984) Biochemistry 23, 944-955; Mirau, P. A., Behling, R. W., & Kearns, D. R. (1985) Biochemistry 24, 6200-6211].     

Selective 31P(1H) nuclear Overhauser effect study on the polar headgroup conformation of phospholipids in micelles in organic solvents

The polar headgroup structure of phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylserine (PS) in inverted micelles in chloroform or benzene was investigated by the selective 31P(H) nuclear Overhauser effect (NOE). In the frequency dependence of the 31P(1H) NOE, PC micelles in CDCl3 showed two maxima. The larger maximum was located at the resonance of the glycerol-CH2OP protons and the smaller at the resonance of the N-methyl protons. In PC/PE mixed micelles in C6D6, both PC and PE showed three maxima which were located at the resonance of the CH2OP protons, the N-methyl protons and the amino protons in the frequency dependence of the 31P-NOE. The N-methyl protons of PC and the amino protons of PE were closely spaced to the phosphate groups of neighboring lipid molecules. The polar headgroups of PC and PE in the mixed micelles were concluded to lie in the plane perpendicular to the molecular axes. The frequency dependence of the 31P(H) NOE for PS micelles in C6D6 showed the maxima at the resonances of the amino protons and the CH2OP protons. The polar headgroups of PS molecules were not extended parallel to the molecular axes in the inverted micelles.     

The task of determining the local structure of proteins by Overhauser effect nuclear spectroscopy addressed once more]

The testing of the earlier developed theoretical method for determining the backbone protein conformations (the local structure) on the basis of the two-dimensional nuclear Overhauser effect (NOE) spectroscopy has been fulfilled. The method approval has been carried out by the calculation (based upon spectral NOE parameters) of the local plastocyanin and bovine pancreatic trypsin inhibitor structures followed by the comparison of the received conformational parameters with the X-ray data. The comparison of the molecular conformations in solution and crystal has been implemented for different fragments of the polypeptide chain (beta-structures, alpha-helices, irregular segments) using the mathematical statistics methods. The verification of the "zero" hypothesis about the similarity of phi and psi variation rows which was carried out at the reliability level of 0.99 showed that in both cases there were no systematic deviations of dihedral angles of the compared conformations and that their dispersion differences were statistically indiscernible. It has been concluded that the approved method permits to determine the local structure of the conformationally rigid proteins (or their fragments) at the level close to that which provides the high resolution X-ray analysis.     

The beta-arrestin pathway-selective type 1A angiotensin receptor (AT1A) agonist [Sar1,Ile4,Ile8]angiotensin II regulates a robust G protein-independent signaling network

The angiotensin II peptide analog [Sar(1),Ile(4),Ile(8)]AngII (SII) is a biased AT(1A) receptor agonist that stimulates receptor phosphorylation, β-arrestin recruitment, receptor internalization, and β-arrestin-dependent ERK1/2 activation without activating heterotrimeric G-proteins. To determine the scope of G-protein-independent AT(1A) receptor signaling, we performed a gel-based phosphoproteomic analysis of AngII and SII-induced signaling in HEK cells stably expressing AT(1A) receptors. A total of 34 differentially phosphorylated proteins were detected, of which 16 were unique to SII and eight to AngII stimulation. MALDI-TOF/TOF mass fingerprinting was employed to identify 24 SII-sensitive phosphoprotein spots, of which three (two peptide inhibitors of protein phosphatase 2A (I1PP2A and I2PP2A) and prostaglandin E synthase 3 (PGES3)) were selected for validation and further study. We found that phosphorylation of I2PP2A was associated with rapid and transient inhibition of a β-arrestin 2-associated pool of protein phosphatase 2A, leading to activation of Akt and increased phosphorylation of glycogen synthase kinase 3β in an arrestin signalsome complex. SII-stimulated PGES3 phosphorylation coincided with an increase in β-arrestin 1-associated PGES3 and an arrestin-dependent increase in cyclooxygenase 1-dependent prostaglandin E(2) synthesis. These findings suggest that AT(1A) receptors regulate a robust G protein-independent signaling network that affects protein phosphorylation and autocrine/paracrine prostaglandin production and that these pathways can be selectively modulated by biased ligands that antagonize G protein activation.     

Interrenal activity in the female lizard Lacerta vivipara J.: in vitro response to ACTH 1-39 and to [Sar1, Val5] angiotensin II (ANG II)

A perifusion system technique was developed in order to determine in vitro the respective roles of ACTH and ANG II in the regulation of adrenal steroidogenesis in the lizard Lacerta vivipara. Synthetic human ACTH 1-39, administered as 20-min pulses, stimulated corticosterone (B) and aldosterone (A) release in a dose-dependent manner. The increase in corticosterone output was higher than that in aldosterone output, leading to an enhancement of the B/A ratio. Iterative stimulations with 1 nM ACTH (20-min pulses every 120 min) led to reproducible increases in corticosterone and aldosterone release. Prolonged stimulation with 1 nM ACTH (up to 240 min) caused a sustained increase in corticosteroid release, suggesting that, in the lizard, ACTH does not induce any desensitization phenomenon. The angiotensin II analogue [Sar1, Val5] ANG II also stimulated corticosterone and aldosterone release in a dose-dependent manner; the stimulatory effects of ANG II on both steroids were very similar. These results indicate that, in lizards, ACTH plays a major role in the regulation of adrenal steroidogenesis. Since ANG II stimulates the production of gluco- and mineralocorticoids, our data raise the question of the existence of two cell types synthesizing corticosterone and aldosterone, respectively, in reptiles.     

A conformational study of alpha-L-Rhap-(1----2)-alpha-L-Rhap-(1----OMe) by NMR nuclear Overhauser effect spectroscopy (NOESY) and molecular dynamics calculations.

The conformational preference of the disaccharide alpha-L-Rhap-(1----2)-alpha-L-Rhap-(1----OMe) (1) about the glycosidic torsion angles, phi and psi, was studied by NMR NOESY spectroscopy and molecular mechanics calculations. The NOE data were consistent with either of two distinct conformations close to minima on a calculated phi/psi potential energy surface. Starting from the lowest energy conformation, a 1-ns molecular dynamics (MD) trajectory was computed in vacuo, from which the NOE curves were simulated and compared to the experimentally observed NOESY data.     

Conformation of the third domain of turkey ovomucoid in solution. Structural analysis by two-dimensional Overhauser nuclear effect spectroscopy]

The conformations of a polypeptide chain of turkey ovomucoid third domain and its modified form with split reactive site peptide bond Leu-18--Glu-19 have been determined by the literary two-dimensional nuclear Overhauser effect spectroscopy data using an earlier suggested method. It has been found that the polypeptide domain backbone contains an alpha-helical fragment (residues 32-47), five segments having extended conformation (1-5, 11-17, 19-25, 29-31, 48-50) and beta-turn type 1 (26-29). Segments 23-26, 28-31 and 50-51 form an antiparallel beta-structure. Conformational states of the residues entering irregular domain segments have been analysed. Splitting of the reactive site peptide bond Leu-18--Glu-19 is shown to cause insignificant changes in the conformations of a number of amino acid residues except for Val-6 and Asp-7 ones which undergo essential conformational alterations. The conformations of domain in solution and of japanese quail ovomucoid third domain in crystal have been compared. The root-mean-square deviations for phi and psi angles indicate their high similarity. The conformations of turkey ovomucoid third domain and proteinase inhibitor BUSI IIA in solution have been analysed. In spite of moderate (50%) homology of primary structures, some 75% of amino acid residues are shown to have close conformational phi and psi parameters.     

125I[Sar(1)Ile(8)] angiotensin II has a different affinity for AT(1) and AT(2) receptor subtypes in ovine tissues

Iodinated angiotensin II (Ang II) and its analogues are often assumed to have equal affinities for AT(1) and AT(2) receptor subtypes. However, using saturation and competition binding assays in several tissues from pregnant, nonpregnant, and fetal sheep, we found the affinity of 125I[Sar(1)Ile(8)] Ang II for Ang II receptors was different (P<0.05) between tissue types. The dissociation constants (Kd) and half maximal displacements of [Sar(1)Ile(8)] Ang II (Sar IC(50)) were directly related (P<0.05) to proportions of AT(1) receptors, and inversely related (P<0.05) to proportions of AT(2) receptors in tissues from all groups combined, in tissues from individual groups (pregnant, nonpregnant or fetal), and in some individual tissues (uterine arteries and aortae). This suggests that 125I[Sar(1)Ile(8)] Ang II has a different affinity for AT(1) and AT(2) receptors in ovine tissues. The Kds of 125I[Sar(1)Ile(8)] Ang II for "pure" populations of AT(1) and AT(2) receptors were 1.2 and 0.3 nM, respectively, i.e. affinity was four-fold higher for AT(2) receptors. We corrected the measured proportions of the receptor subtypes using their fractional occupancies. In tissues which contained at least 10% of each receptor subtype, the corrected proportions were significantly altered (P<0.05), even in some tissues, to the extent of being reversed.     

19F]-1H heteronuclear nuclear Overhauser effect studies of the acyl chain-binding site of acyl carrier protein

[19F]-1H heteronuclear difference nuclear Overhauser effect experiments are performed on a sample of 5,5-difluorohexanoyl acyl carrier protein from Escherichia coli. Interaction of the fluorines at the 5-position of the acyl chain with protons on methyl groups of isoleucine 54 and alanine 59 is clearly indicated. The covalent attachment of the acyl chain via a prosthetic group to serine 36 and the known alpha-helix which exists from residues 36 to 51 greatly restrict the structural models which would allow acyl chain contact with residues 54 and 59.     

Sar1Ile7]angiotensin III, a new selective antagonist of the pressor effect of angiotensin III in conscious rats

Two analogues of angiotensin III were compared as antagonists of the pressor response to angiotensin II (ANG II) and angiotensin III (ANG III) in conscious, unrestrained rats. Dose-mean arterial pressure (MAP) response curves were obtained for ANG II and ANG III in the absence or presence of [Ile7]ANG III (1.3 x 10(-7) mol/kg) or [Sar1 Ile7]ANG III (1.2 x 10(-7) mol/kg). In the presence of [Ile7]ANG III, the dose-MAP response curves for ANG II and ANG III were significantly displaced to the right. [Ile7]ANG III behaved as a partial agonist on ANG II but not ANG III receptors. In the presence of [Sar1 Ile7]ANG III, the dose-MAP response curve for ANG III but not ANG II was significantly displaced to the right. This suggests that [Sar1 Ile7]ANG III is a selective antagonist of ANG III in the vasculature. [Ile7]ANG III, on the other hand, antagonizes both ANG II and ANG III receptors. Our results support the hypothesis of the existence of a sub-class of angiotensin receptors activated by ANG III in the vascular smooth muscle.     

Structure in the polar head region of phospholipid bilayers: A 31P [1H] nuclear Overhauser effect study.

The structure of the head-group region of some phospholipid bilayers in vesicle form has been studied and an intermolecular association of the N-methyl protons of phosphatidylcholine (PC) with the phosphate of phosphatidylethanolamine (PE) in mixed vesicles has been identified. Observation of a 31P[1H] nuclear Overhauser effect (NOE) in the phosphorus nuclear magnetic resonances of both PC and PE in mixed vesicles demonstrates an intimate dipolar interaction between some protons and the phosphorus nuclei. Substitution of deuterium for the N-methyl protons of PC eliminated the majority of the effect and necessitated the construction of a model of the bilayer surface in which the N-methyl protons of PC could interact closely with the phosphates of neighboring PE molecules. The predominant orientation of the head group must then be parallel to the bilayer surface. The amino protons of PE do not contribute significantly to the observed NOE. A corollary of these results is that there is little if any tendency for either PC or PE in the mixed vesicles to segregate into separate domains. A decrease in NOE in sphingomyelin vesicles on going from H2O to D2O suggests that an exchangeable proton contributes to the NOE. In addition the low value of the NOE observed in D2O suggests that the head-group conformation of sphingomyelin differs from that of PC.     

Conformation of the oligonucleotide d(AAAAAATTTTTT)2 by two-dimensional nuclear Overhauser effect spectroscopy and its relevance to poly(dA).poly(dT)

Conformational analysis from the pattern and intensities of cross-peaks in the two-dimensional nuclear Overhauser effect proton nmr spectra of the homopolymer, poly(dA) · poly(dT), and the analogous oligomer, d(AAAAAATTTTTT)₂, indicate that they both exist in the B-conformation. The conformation of the ApT/TpA junction in the oligomer is significantly different from the rest of the base pairs.