A novel β-thymosin from the sea urchin: extending the phylogenetic distribution of β-thymosins from mammals to echinoderms

The study of the phylogenetic distribution of the β-thymosin family is important to elucidate its biological function further. A new thymosin, designated as thymosin β₁₄, consisting of 40 amino acid residues and with a molecular weight of 4537 Da as determined by ion spray mass spectrometry, was isolated from the sea urchin. The N-terminus of this polypeptide is blocked by an acetyl group as found by matrix-assisted laser desorption mass spectrometric and amino acid analysis. The primary structure was elucidatd by Edman degradation of the HPLC-purified thymosin β₁₄ fragments produced by digestion with endoproteinase Asp-N and trypsin. Sequence comparison reveals that thymosin β₁₄ is 73% homologous to thymosin β₄, obtained from calf thymus. By isolating and characterising the structure of thymosin β₁₄ from the sea urchin, an invertebrate, substantial knowledge about the phylogenetic distribution and evolution of β-thymosins is gained. © 1997 European Peptide Society and John Wiley & Sons Ltd.     

Role of two consecutive α,β-dehydrophenylalanines in peptide structure: Crystal and molecular structure of Boc-Leu-ΔPhe-ΔPhe-Ala-Phe-NHMe

An N^α-protected model pentapeptide containing two consecutive ΔPhe residues, Boc-Leu-ΔPhe-ΔPhe-Ala-Phe-NHMe, has been synthesized by solution methods and fully characterized. ¹H-nmr studies provided evidence for the occurrence of a significant population of a conformer having three consecutive, intramolecularly H-bonded β-bends in solution. The solid state structure has been determined by x-ray diffraction methods. The crystals grown from aqueous methanol are orthorhombic, space group P2₁2₁2₁, a = 11.503(2), b = 16.554(2), c = 22.107(3) Å, V = 4209(1) Å,³ and Z = 4. The x-ray data were collected on a CAD4 diffractometer using CuK_a radiation (λ = 1.5418 Å). The structure was determined using direct methods and refined by full-matrix least-squares procedure. The R factor is 5.3%. The molecule is characterized by a right handed 3₁₀-helical conformation (〈ϕ〉 = −68.2°, 〈ψ〉 = −26.3°), which is made up of two consecutive type III β-bends and one type I β-bend. In the solid state the helical molecules are aligned head-to-tail, thus forming long rod like structures. A comparison with other peptide structures containing consecutive ΔPhe residues is also provided. The present study confirms that the -ΔPhe-ΔPhe-sequence can be accommodated in helical structures. © 1997 John Wiley & Sons, Inc. Biopoly 42: 373–382, 1997     

A CD and an nmr study of multiple bradykinin conformations in aqueous trifluoroethanol solutions

CD and nmr studies have been carried out on aqueous trifluoroethanol (TFE) solutions of bradykinin (BK) and a bradykinin antagonist. The CD results exhibit a striking effect of TFE on the spectra of BK, with sequence Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg, and the BK antagonist, with sequence D -Arg-Arg-Pro-Hyp-Gly-Thi-D -Ser-D -Cpg-Cpg-Arg [where Hyp is 4-hydroxy-L -proline; Thi refers to β-(2-thienyl)-L -alanine and Cpg refers to α-cyclopentylglycine]. The effect of increasing concentration of TFE in water on the difference ellipticity at 222 nm was examined and showed that BK may be a mixture of at least two different conformers, one of which largely forms when the TFE concentration is increased beyond 80%. The linear extrapolation of 100% of the difference ellipticity of BK at low TFE concentrations yields a value in agreement with that shown by the BK antagonist, indicating that the conformation of BK at the lower TFE concentrations is similar to that of the BK antagonist. The conformational analysis was carried out using both one-dimensional and two-dimensional ¹H-nmr techniques. The total correlation spectroscopy (TOCSY) spectrum of BK in a 60/40% (v/v) TFE/H₂O solution at 10°C and a nuclear Overhauser effect spectroscopy (NOESY) spectrum that shows only sequential H_α(i) – NH(i + 1) or the H_α(i) – H_δδ′(i + 1) NOEs indicate that the majority of the molecules adopt an all-trans extended conformation. The TOCSY for BK in the 95/5% (v/v) TFE/H₂O solution shows that there are two major conformations in the solution with about equal population. The NOESY experiment shows two new important cross peaks for one conformation, namely Pro²(α)-Pro³ (α) and the Pro²(α)-Gly⁴(NH), indicating a cis Pro²-Pro³ bond and a type VI β-turn between residues Arg¹ and Gly⁴ involving cis proline at position 3, respectively. The low temperature coefficient of Gly⁴ for this conformation suggests the presence of an intramolecular hydrogen bond, therefore a type VIa β-turn is present. The other conformation is all trans and extended. The BK antafonist shows difference CD spectra in TFE solutions referred to H₂O that are superficially indicative of a β-bend. However, nmr speaks against this possibility, as only one set of peaks were observed in the TOCSY and NOESY experiments, indicating an all-trans extended confirmation over the range of TFE concentrations. The BK-antagonist CD data suggest that solvent perturbation of the CD of an extended confirmation perturbation of the optical activity of the thienyl moiety of the peptide since the CD spectrum of N-acetyl-β-thienyl-L -alanine N-methylamide is strongly perturbed by TFE. The present results again demonstrate the complementary relationship between CD and nmr. © 1994 John Wiley & Sons, Inc.     

A pentapeptide model for an early folding step in the refolding of staphylococcal nuclease: The role of its turn propensity

Recently the folding of a staphylococcal nuclease (P117G) variant was examined with the hydrogen-deuterium (H-D) exchange technique. Many of the residues that showed significant protection are located in protection are located in β-sheet regions. About half the residues protected belong to an antiparallel β-hairpin structure (residues 21–35) in the native structure. The β-hairpin structure is formed by strands 2 and 3 of sheet 2 connected by the sequence²⁷ Y KGQP³¹ in a type I′ reverse turn conformation with a 4 → 1 hydrogen bonding between Q30 NH and Y27 C=O. We have targeted the conformational characterization of the peptide model Ac-YKGQP-NH₂ with ¹II two-dimensional nmr techniques in aqueous solution with a view to assessing its propensity to sample turn conformational forms and thus initiate the formation of β-hairpin structure. Based upon the observed dαn (i, i + 1), dαn (i, i + 3), and dnn (i, i + 1) nuclear Overhauser effect connectivities, temperature coefficients for amide protons and conformational analysis with quantum mechanical perturbative configuration interaction over localized orbitals method, we conclude that the model peptide samples turn conformational forms with reduced conformational entropy. We suggest that the turn can nucleate the formation of the β-hairpin structure in the refolding of nuclease. Observation of turn propensity for this sequence is consistent with the folding mechanism of the Greek key motif (present in Staphylococcal nuclease) proposed in the literature. © 1997 John Wiley & Sons, Inc.     

Thymosin β4 and Tissue Transglutaminase. Molecular Characterization of Cyclic Thymosin β4

Thymosin β₄ is the prototype of β-thymosins and is present in almost every mammalian cell. It is regarded to be the main intracellular G-actin sequestering peptide. Thymosin β₄ serves as a specific glutaminyl substrate for guinea pig transglutaminase. In the absence of an appropriate additional aminyl donor an ε-amino group of thymosin β₄ serves also as an aminyl substrate and an intramolecular bond is formed concomitantly NH₃ (17 Da) is lost. The molecular mass of the product is 4,949.6 Da. This is 16.3 Da less than the molecular mass of thymosin β₄ (4,965.9 Da). Digestion with endopeptidases and Edman degradation of the fragments identified the exact position of the ring forming isopeptide bond. In spite of 3 glutaminyl and 9 lysyl residues of thymosin β₄ only one isopeptide bond between Lys16 and Gln36 was formed (cyclic thymosin β₄). These two amino acid residues are conserved in all β-thymosins. Cyclic thymosin β₄ still forms a complex with G-actin albeit the stability of the complex is about one fiftieth of the stability of the thymosin β₄ × G-actin complex.     

Determination of the molecular conformation of beta-D-O2,2'-cyclouridine in aqueous solution by proton magnetic resonance spectroscopy

The solution conformation of β-D -O²,2′-cyclouridine has been determined at 27 and 88°C in D₂O by proton magnetic resonance spectroscopy. The conformation is described in terms of a fixed syn-like sugar-base torsional angle, a type S furanose ring conformation (similar to 2′-endo), and a temperature-dependent exocyclic C(4)′–C(5′) rotamer population containing approximately 50% of the gauche-gauche form at 27°C. β-D -O²,2′-Cyclouridine 5′-phosphate likewise possesses a type S furanose ring conformation.     

Type II β-turn conformation of pivaloyl-L-prolyl-α-aminoisobutyryl-N-methylamide: Theoretical,spectroscopic, and X-ray studies

Pivaloyl-L -Pro-Aib-N-methylamide has been shown to possess one intramolecular hydrogen bond in (CD₃)₂SO solution, by ¹H-nmr methods, suggesting the existence of β-turns, with Pro-Aib as the corner residues. Theoretical conformational analysis suggests that Type II β-turn conformations are about 2 kcal mol^?1 more stable than Type III structures. A crystallographic study has established the Type II β-turn in the solid state. The molecule crystallizes in the space group P2₁ with a = 5.865 Å, b = 11.421 Å, c = 12.966 Å, β = 97.55°, and Z = 2. The structure has been refined to a final R value of 0.061. The Type II β-turn conformation is stabilized by an intramolecular 4 → 1 hydrogen bond between the methylamide NH and the pivaloyl CO group. The conformational angles are ?_Pro = ?57.8°, ψ_Pro = 139.3°, ?_Aib = 61.4°, and ψ_Aib = 25.1°. The Type II β-turn conformation for Pro-Aib in this peptide is compared with the Type III structures observed for the same segment in larger peptides.     

Folded Structures in Protonated Reduced Dipeptides

Reduced dipeptides with the general formula RCO-Xaa- rXbb-N⁺HR′R′′ (rXbb, reduced analogue of residue Xbb: NH-C^α HR¹ -C_r H₂) are shown to adopt a folded conformation in solution and in the solid state. The protonated reduced amide bond is an active proton donor capable of interacting with a peptide carbonyl to give a strong hydrogen bond topologically equivalent to the i+2 or i+3? i interaction. The resulting conformation is similar to the γ- or β-turn structure found in peptides and proteins.     

Conformational characterization of peptides rich in the cycloaliphatic Cα,α-disubstituted glycine 1-amino-cyclononane-1-carboxylic acid

A series of N- and C-protected, monodispersed homo-oligopeptides (to the pentamer level) from the cycloaliphatic C^α,α,-dialkylated glycine 1-aminocyclononane-1-carboxylic acid (Ac₉c) and two Ala/Ac₉c tripeptides have been synthesized by solution methods and fully characterized. The conformational preferences of all the model peptides were determined in deuterochloroform solution by FT-IR absorption and ¹H-NMR. The molecular structures of the amino acid derivatives mClAc-Ac₉c-OH and Z-Ac₉c-OtBu, the dipeptide pBrBz-(Ac₉c)₂-OtBu, the tetrapeptide Z-(Ac₉c)₄-OtBu, and the pentapeptide Z-( Ac₉c)₅-OtBu were determined in the crystal state by X-ray diffraction. Based on this information, the average geometry and the preferred conformation for the cyclononyl moiety of the Ac₉c residue have been assessed. The backbone conformational data are strongly in favour of the conclusion that the Ac₉c residue is a strong β-turn and helix former. A comparison with the structural propensity of α-aminoisobutyric acid, the prototype of C^α,α-dialkylated glycines, and the other extensively investigated members of the family of 1-aminocycloalkane-1-carboxylic acids (Ac_nc, with n=3−8) is made and the implications for the use of the Ac₉c residue in conformationally constrained analogues of bioactive peptides are briefly examined. © 1997 European Peptide Society and John Wiley & Sons, Ltd. J. Pep. Sci. 3: 367–382 No. of Figures: 10. No. of Tables: 6. No. of References: 62     

Design of Peptides Using α,β-Dehydro-residues: Synthesis,Crystal Structure and Molecular Conformation of N-Boc-L-Val-ΔPhe–ΔPhe-L-Ala-OCH3

To obtain general rules of peptide design using α,β-dehydro-residues, a sequence with two consecutive ΔPhe-residues, Boc-L -Val-ΔPhe–ΔPhe- L -Ala-OCH₃, was synthesized by azlactone method in solution phase. The peptide was crystallized from its solution in an acetone/water mixture (70:30) in space group P6₁ with a=b=14.912(3) Å, c= 25.548(5) Å, V=4912.0(6) ų. The structure was determined by direct methods and refined by a full matrix least-squares procedure to an R value of 0.079 for 2891 observed [I?3σ(I)] reflections. The backbone torsion angles ?₁=?54(1)°, ψ₁= 129(1)°, ω₁=?177(1)°, ?₂ =57(1)°, ψ₂=15(1)°, ω₂ =?170(1)°, ?₃=80(1)°, ψ₃ =7(2)°, ω₃=?177(1)°, ?₄ =?108(1)° and ψ^T₄=?34 (1)° suggest that the peptide adopts a folded conformation with two overlapping β-turns of types II and III′. These turns are stabilized by two intramolecular hydrogen bonds between the CO of the Boc group and the NH of ΔPhe³ and the CO of Val¹ and the NH of Ala⁴. The torsion angles of ΔPhe² and ΔPhe³ side chains are similar and indicate that the two ΔPhe residues are essentially planar. The folded molecules form head-to- tail intermolecular hydrogen bonds giving rise to continuous helical columns which run parallel to the c-axis. This structure established the formation of two β-turns of types II and III′ respectively for sequences containing two consecutive ΔPhe residues at (i+2) and (i+3) positions with a branched β-carbon residue at one end of the tetrapeptide.     

Reverse Relationship between α-Carbon Chirality and Helix Handedness in (αMe)Phe Peptides

Abstract

The crystal-state preferred conformations of two tripeptides, one tetrapeptide, and one pen- tapeptide, each containing a single residue of the chiral, C^α,α-disubstituted glycine C^α-methyl, C^α-benzylglycine [(αMe)Phe], have been determined by X-ray diffraction. The tripeptides are Z-L-(αMe)Phe-(Aib)₂-OH dihydrate and Z-Aib-D-(αMe)Phe-Aib-OtBu, the tetrapeptide is Z-(Aib)₂-D-(αMe)Phe-Aib-OtBu, and the pentapeptide is pBrBz-(Aib)₂-DL-(αMe)Phe-(Aib)₂-OtBu. While the two tripeptides are folded in a β-bend conformation, two such conformations are consecutively formed by the tetrapeptide. The pentapeptide adopts a regular 3₁₀-helix promoted by three consecutive β-bends. This study confirms the strong propensity of short peptides containing C^α-methylated α-aminoacids to fold into β-bends and 3₁₀-helical structures. Since Aib is achiral, the handedness of the observed bends and helices is dictated by the presence of the (αMe)Phe residue. In general, we have found that the relationship between (αMe)Phe chirality and helix handedness is opposite to that exhibited by protein aminoacids. A comparison with the preferred conformation of other extensively investigated C^α-methylated aminoacids is made.     

Conformation of sequential polypeptides of (Lysi-Leuj), (Lysi-Serj), and (Lys-Gly) in sodium dodecyl sulfate solution

A series of sequential polypeptides (Lys_iR_j)_n (R is Leu, Ser, or Gly) and random copolypeptides, (Lys^x, Leu^y)_n, were synthesized. Their conformation in NaDodSO₄ solution was determined by CD. Only (Lys-Leu)_n, (Lys-Ser)_n, and (Lys₃-Ser)_n adopt a stable β-form in the surfactant solution; (Lys-Ser₂)_n, (Lys-Ser₃)_n, (Lys₂-Ser₂)_n, and (Lys₂-Ser)_n have an unstable β-form, which reverts to an unordered form in high NaDodSO₄ concentrations, even though both Ser and DodSO-bound Lys⁺ are β-formers. In contrast, (Lys-Gly)_n remains unordered in NaDodSO₄ solution. On the other hand, Lys-rich (Lys₂-Leu)_n forms an unstable helix and (Lys₂-Leu₂)_n a stable helix in NaDodSO₄ solution. In 25 mM NaDodSO₄ (Lys^x, Leu^y)_n also forms a helix up to x = 75 and reverts to the β-form at x = 90. This compares with the helical conformation of (Lys^x, Ala^y)_n up to x = 65 and its β-form at x = 90, suggesting that Leu is an even stronger helix-former than Ala. Our results may provide a plausible explanation for the increase in helicity and disruption of the β-form for many proteins in NaDodSO₄ solution, that is, the polypeptide chain of a protein usually favors a helical conformation over a β-form in the presence of excess surfactant.     

Conformational and binding properties of chicken liver basic fatty acid binding protein in solution

The conformation of basic fatty acid binding protein from chicken liver and the binding properties of the apo protein toward 11-dansylamino-undecanoic acid were investigated by CD and fluorescence spectroscopy. In one set of experiments the binding process was followed by the appearance of induced optical activity in the absorption region of the dansyl chromophore. In a second set of experiments the binding process was followed by the large enhancement of emission fluorescence of the dansyl fluorophore. From the saturation curves, the stoichiometry of the complex and the binding constant of the fatty acid to the protein were precisely determined. The values of the dissociation constant determined with the two methods were in excellent agreement: we obtained K_D = (1.0 ± 0.1) · 10^?6M in a 0.9 : 1 stoichiometry. The native conformation of the protein is remarkably stable in a variety of solvent systems, including acetonitrile–water, ethylene glycol–water, and dicxane–water of various compositions. The CD results also showed that the binding of the fatty acid does not induce any appreciable change in the protein conformation. In a mixture of water and 2,2,2-trifluoroethanol 1 : 9 (v/v), the native conformation collapses and a new ordered structure is formed, characterized by a high amount of α-helix. © 1994 John Wiley & Sons, Inc.     

NMR evidence for a type I β-turn in (Pro2)-tetrapeptides and interdependence of cis:Trans isomerism,ring flexibility,and backbone conformation

Tetrapeptides with proline in position 2, asparagine or leucine in position 3, and glycine in positions 1 and 4, with end groups free or blocked on the N-terminal side, were studied in their various ionic states in ²H₂O and in Me₂SO-d₆ by ¹H- and ¹³C-nmr. In order to clarify or refine some details, successive substitutions of the residues in these peptides with amino acids enriched to 85% in ¹³C, or to 85% ¹³C plus 97% ²H were carried out. The ¹H and ¹³C chemical shifts as well as the ¹H-¹H, ¹³C-¹³C, and ¹³C-¹H coupling constants and the signal intensities show strong similarity of behavior between the tetrapeptides of asparagine and leucine. The main conformational characteristics are (1) the almost total stabilization of the trans conformer in the type I β-turn structure when the peptide is in the zwitterion state dissolved in Me₂SO. This is deduced from the ³J and the ³J coupling constants, which both furnish a dihedral angle of ?³ = ?90°, and from the positive value of the temperature coefficient of the glycine-4 amide protons, which suggests a type 4 → 1 hydrogen bond; (2) the evolution of cis and trans isomer fractions which change with the ionic state of the peptides in Me₂SO, whereas they remain constant in aqueous solution; and (3) the conformation of the pyrrolidine ring as it follows the variations in cis:trans isomer populations together with the side-chain rotamer fractions of the residue in position 3. In the β-turn conformation the isomer cis is less abundant and the pyrrolidine ring is more flexible; this explains the perfect accommodation of the proline residue in position 2 of a bend. The interdependence of these phenomena where interactive forces play a predominant role underlines the importance of cooperative effects in the molecule. The results also suggest that the cis isomer of proline can adapt itself just as well as the trans isomer to position 2 of a type I β-turn.     

Thymosin β11: A peptide from trout liver homologous to thymosin β4

A peptide containing 41 amino acid residues has been isolated from trout liver and identified as a member of the β-thymosin family. Sequence analysis shows it to be 78% homologous to thymosin β₄, which is the peptide present in the thymus and other tissues of higher vertebrates, including, as reported here, livers of a species of reptile. Thymosin β₁₁ appears to replace the more prevalent thymosin β₄ in at least two species of bony fish, and represents the sixth structurally characterized member of this widely distributed family of peptides.     

NMR structure of human thymosin alpha-1

800 MHz NMR structure of the 28-residue peptide thymosin alpha-1 in 40% TFE/60% water (v/v) has been determined. Restrained molecular dynamic simulations with an explicit solvent box containing 40% TFE/60% TIP3P water (v/v) were used, in order to get the 3D model of the NMR structure. We found that the peptide adopts a structured conformation having two stable regions: an alpha-helix region from residues 14 to 26 and two double β-turns in the N-terminal twelve residues which form a distorted helical structure.     

Conformational interconversions in peptide β-turns: Discrimination between enantiomeric conformations by chiral perturbation

The crystal structure of the model tripeptide Boc-Aib-Gly-Leu-OMe ( 1 ) reveals two independent molecules in the asymmetric unit that adopt “enantiomeric” type I and type I′ β-turn conformations with the Aib and Gly residues occupying the corner (i + 1 and i + 2) positions. ¹³C cross polarization and magic angle sample spinning spectra in the solid state also support the coexistence of two conformational species. ¹³C-nmr in CDCl₃ establishes the presence of a single species or rapid exchange between conformations. 400 MHz ¹H-nmr provides evidence for conformational exchange involving a major and minor species, with β-turn conformations supported by the low solvent exposure of Leu(3) NH and the observation of N_iH ↔ N_i+1H nuclear Overhauser effects. CD bands in the region 190–230 nm are positive, supporting a major population of type I′ β-turns. The isomeric peptide, Boc-Gly-Leu-Aib-OMe ( 2 ), adopts an “open” type II′ β-turn conformation in crystals. Solid state and solution nmr support population of a single conformational species. Chiral perturbation introduced outside the folded region of peptides may provide a means of modulating screw sense in achiral sequences. © 1998 John Wiley & Sons, Inc. Biopoly 45: 191–202, 1998     

A 1H and 13C NMR Study on the Role of Salt-Bridges in the Formation of a Type I β-Turn in N-Acetyl-L-Asp-L-Glu-L-Lys-L-Ser-NH2

Abstract

The conformation of the tetrapeptide N-Acetyl-Asp⁷-Glu⁸-Lys⁹-Ser¹⁰-NH₂, a fragment of the type I collagen α-1 chain N-telopeptide, has been studied by ¹H and ¹³C NMR and circular dichroism spectroscopy. The spectroscopic evidence, based on two-dimensional, phase-sensitive NMR techniques such as COSY, ROESY, proton-carbon shift correlation and selective COLOC, indicates a strong dependence of the conformation on the experimental conditions. In CD₃OH/H₂O (60/40) at ca. neutral pH the tetrapeptide forms a β-turn, stabilized by a hydrogen bond between NH(S¹⁰) and CO(D⁷) and a strong salt-bridge between COO^?(E⁸) and NH₃ ⁺(K⁹). The β-turn is type I and appears to coexist with a non-hydrogen-bonded structure. The coexistence of these two conformers is proven by proton NMR data such as NH-NH ROEs, reduced NH-H_α(E⁸) coupling constant NH(E⁸) low-field shift and the temperature coefficient of NH(S¹⁰), whereas the conclusion regarding the salt-bridge is based on ¹³C results. In the same solvent, at a pH below the pKa of the carboxyl groups, no evidence for a conformation other than extended can be found. In aqueous solution at approximately neutral pH, evidence for the E⁸-K⁹ charge interaction is observed, but not for a hydrogen bond anywhere in the molecule.     

Structural versatility of peptides from Cα,α-disubstituted glycines: Preferred conformation of the chiral isovaline residue

The molecular structures of four protected isovaline- (Iva-) containing peptides to the pentamer level have been determined by x-ray diffraction. The peptides are t-Boc-Ala-(S)-Iva-Ala-OMe (t-Boc : tert-butyloxycarbonyl; OMe : methoxy) and its (R)-Iva diastereomer, and t-Boc-[Ala-(R)-Iva]₂-Ala-OH and its (S)-Iva diastereomeric methyl ester analogue. The two tripeptides are folded in an open type II β-bend conformation. The fully developed right-handed 3₁₀-helix formed by the (R)-Iva pentapeptide, which includes an unusual intramolecular (acid) O? H ?O?C(peptide) H bond, is partially unfolded (near the C-terminus) in the (S) -Iva pentapeptide. ¹H-nmr and Fourier transform ir absorption studies suggest that in CDCl₃ solution (a) the two tripeptides maintain a type II β-bend conformation of comparable stability and (b) both diastereomeric pentapeptide sequences adopt a fully developed 3₁₀-helix. A comparison with the preferred conformation of other extensively investigated C^α,α-disubstituted glycines is made and the implications for the use of the Iva residue in designing conformationally constrained analogues of bioactive peptides are briefly discussed.