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.     

310-Helices,Helix Screw Sense and Screw Sense Reversal in the Dehydro-peptide Boc-Val-ΔPhe-Gly-ΔPhe-Val-OMe

The pentapeptide Boc-Val-ΔPhe-Gly-ΔPhe-Val-OMe, containing two dehydro-phenylalanine (ΔPhe) residues, has been synthesized and its structure investigated. In the crystalline state, the molecule adopts a right-handed 3₁₀-helical conformation stabilized by two intramolecular hydrogen bonds between CO of Val¹ and NH of ΔPhe⁴, and between CO of ΔPhe² and NH of Val⁵, respectively. NMR measurements are consistent with the presence of 3₁₀-helical structures also in acetonitrile and dimethylsulphoxide solution: the distances between backbone protons estimated from NOE connectivities are in overall agreement with those observed in the solid state; the chemical shifts of the amide protons show the smaller temperature coefficients for the NHs that in solid state are involved in intramolecular hydrogen bonds. The CD spectra in acetonitrile, chloroform, methanol and dimethylsulphoxide display exciton couplets of bands corresponding to the ΔPhe electronic transition at 280nm; the sign of the bands is consistent with the presence of helical structures having a prevalent left-handed screw sense. Addition of 1,1,1,3,3,3-hexafluoro- propan-2-ol gives rise to the gradual appearance of a couplet of opposite sign, suggesting the helix reversal from left-handed sense to right-handed sense. The conformational behaviour is discussed on the basis of the specific sequence of the peptide.     

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 search for the ideal type I β-turn

In 1968 C. Venkatachalam (Biopolymers, Vol. 6, pp. 1425–1436) predicted the ideal forms of β-turns (type I, type II, etc.) based entirely on theoretical calculations. Subsequently, over a thousand x-ray structures of different globular proteins have been analyzed, with results suggesting that the most important form among the hairpin conformers is the type I β-turn. For the latter type of hairpin conformation, the original computations had predicted ϕ_i+1 = −60°, ψ_i+1 = −30°, ϕ_i+2 = −90°, and ψ_i+2 = 0° as backbone torsion angle values, and these have been used from that time as reference values for the identification of the type I β-turn. However, it has never been clarified whether these “ideal” backbone torsion angle values exist in real structures, or whether these torsion angles are only “theoretical values.” Using the most recent release of the Protein Data Bank (1994), a survey has been made to assign amino acid pairs that approach the ideal form of the type I β-turn. The analysis resulted in four sequences where the deviation from ideal values for any main-chain torsion angles was less than 2°. In order to determine whether such a backbone fold is possible only in proteins owing to fortuitous cooperation of different folding effects, or whether it occurs even in short peptides, various attempts have been made to design the optimal amino acid sequence. Such a peptide model compound adopting precisely the predicted torsion angle values [ϕ_i+1 = −60°, ψ_i+1 = −30°, ϕ_i+2 = −90°, and ψ_i+2 = 0°] could provide valuable information. The solid state conformation of cyclo[(δ) Ava-Gly-Pro-Thr (O¹Bu)-Gly] reported herein, incorporating the -Pro-Thr- subunit, yields values suggesting that the “ideal” type I β-turn is even possible for a peptide where there are no major environmental effects present. © 1996 John Wiley & Sons, Inc.     

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.     

Crystal and molecular structure of benzyloxycarbonyl-α-aminoisobutyryl-L-prolyl methylamide: The observation of an X2-Pro3 Type III β-Turn

The crystal and molecular structure of N-benzyloxycarbonyl-α-aminoisobutyryl-L -prolyl methylamide, the amino terminal dipeptide fragment of alamethicin, has been determined using direct methods. The compound crystallizes in the orthorhombic system with the space group P2₁2₁2₁. Cell dimensions are a = 7.705 Å, b = 11.365 Å, and c = 21.904 Å. The structure has been refined using conventional procedures to a final R factor of 0.054. The molecular structure possesses a 4 → 1 intramolecular N-H—O hydrogen bond formed between the CO group of the urethane moiety and the NH group of the methylamide function. The peptide backbone adopts the type III β-turn conformation, with ?₂ = ?51.0°, ψ₂ = ?39.7°, ?₃ = ?65.0°, ψ₃ = ?25.4°. An unusual feature is the occurrence of the proline residue at position 3 of the β-turn. The observed structure supports the view that Aib residues initiate the formation of type III β-turn conformations. The pyrrolidine ring is puckered in C^γ-exo fashion.     

β-Turn conformations in crystal structures of model peptides containing α,α-Di-n-propylglycine and α,α-Di-n-butylglycine

The crystal state conformations of three peptides containing the α,α-dialkylated residues. α,α-di-n-propylglycine (Dpg) and α,α-di-n-butylglycine (Dbg), have been established by x-ray diffraction. Boc-Ala-Dpg-Alu-OMe (I) and Boc-Ala-Dbg-Ala-OMe (III) adopt distorted type II β-turn conformations with Ala (1) and Dpg/Dbg (2) as the corner residues. In both peptides the conformational angles at the Dxg residue (I: ? = 66.2°, ψ = 19.3°; III: ? = 66.5°. ψ = 21.1°) deviate appreciably from ideal values for the i + 2 residue in a type II β-turn. In both peptides the observed (N…O) distances between the Boc CO and Ala (3) NH groups are far too long (1: 3.44 Å: III: 3.63 Å) for an intramolecular 4 → 1 hydrogen bond. Boc-Ala-Dpg-Ata-NHMe (II) crystallizes with two independent molecules in the asymmetric unit. Both molecules HA and HB adopt consecutive β-turn (type III-III in HA and type III-I in IIB) or incipient 3₁₀-helical structures, stabilized by two intramolecular 4 → 1 hydrogen bonds. In all four molecules the bond angle N-C^α-C′ (τ) at the Dxg residues are ≥ 110°. The observation of conformational angles in the helical region of ?,ψ space at these residues is consistent with theoretical predictions. © 1995 John Wiley & Sons, Inc.     

Synthesis and conformational study of peptides possessing helically arranged ΔZPhe side chains

Z-Dehydrophenylalanine (Δ^zPhe) possessing four oligopeptides, Boc-(L -Ala-Δ^zPhe-Aib)_n-OCH₃ (n = 1–4: Boc, t-butoxycarbonyl; Aib, α-aminoisobutyric acid), were synthesized, and their solution conformations were investigated by ¹H-nmr, ir, uv, and CD spectroscopy and theoretical CD calculation. ¹H-nmr (the solvent accessibility of NH groups) and ir studies indicated that all the NH groups except for those belonging to the N-terminal L -Ala-Δ^zPhe moiety participate in intramolecular hydrogen bonding in chloroform. This suggests that the peptides n = 2–4 have a 4 → 1 hydrogen-bonding pattern characteristic of 3₁₀-helical structures. The uv spectra of all these peptides recorded in chloroform and in trimethyl phosphate showed an intense maximum around 276 nm assigned to the Δ^zPhe chromophores. The corresponding CD spectra of the peptides n = 2–4 showed exciton couplets with a negative peak at longer wavelengths, whereas that of the peptide n = 1 showed only weak signals. Theoretical CD spectra were calculated for the peptides n = 2–4 of several helical conformations, on the basis of exciton chirality method. This calculation indicated that the three peptides form a helical conformation deviating from the perfect 3₁₀-helix that contains three residues per turn, and that their side chains of Δ^z Phe residues are arranged regularly along the helix. The center-to-center distance between the nearest phenyl pair(s) was estimated to be ～ 5.5 Å. The chemical shifts of the Δ^zPhe side-chain protons (H^β and aromatic H) for the peptides n = 2–4 indicated anisotropic shielding effect of neighboring phenyl group(s); the effect also supports a regular arrangement of the Δ^z Phe side chains along the helical axis. © 1993 John Wiley & Sons, Inc.     

Synthesis,and crystal and molecular structure of the 310-helical α,β-dehydro pentapeptide Boc-Leu-Phe-Ala-ΔPhe-Leu-Ome

α,β-Dehydro amino acid residues are known to constrain the peptide backbone to the β-bend conformation. A pentapeptide containing only one α,β dehydrophenylalanine (ΔPhe) residue has been synthesized and crystallized, and its solid state conformation has been determined. The pentapeptide Boc-Leu-Phe-Ala-ΔPhe-Leu-OMe (C₃₉H₅₅N₅O₈, M_w = 721.9) was crystallized from aqueous methanol. Monoclinic space group was P2₁, a = 10.290(2)°, b = 17.149(2)°, c = 12.179(2) Å, β = 96.64(1)° with two molecules in the unit cell. The x-ray (Mo K_α, λ = 0.7107A) intensity data were collected using a CAD4 diffractometer. The crystal structure was determined by direct methods and refined using least-squares technique. R = 4.4% and R_w = 5.4% for 4403 reflections having |F₀| ≥ 3σ(|F₀|). All the peptide links are trans and the pentapeptide molecule assumes 3₁₀-helical conformation. The mean ?,ψ values, averaged over the first four residues, are ?64.4°, ?22.4° respectively. There are three 4 → 1 intramolecular hydrogen bonds, characteristic of 3₁₀,-helix. In the crystal, the peptide helices interact through two head-to-tail. N? H? O intermolecular hydrogen bonds. The peptide molecules related by 2₁, screw symmetry form a skewed assembly of helices. © 1995 John Wiley & Sons, Inc.     

Conformational behaviour of Cα,α-diphenylglycine: folded vs. extended structures in DϕG-containing tripeptides

The crystal structures of three fully protected tripeptides containing the Dϕg residue (C^α,α-diphenylglycine) in the central position are reported, namely Z-Gly-Dϕg-Gly-OMe ( a ), Z-Gly-Dϕg-Aib-OMe ( b ) and Z-Aib-Dϕg-Aib-OMe ( c ). The molecular conformations are quite unusual because the Dϕg residue adopts a folded conformation in the 3₁₀-helical region when the following residue adopts a folded conformation of opposite handedness (peptides b and c ). In contrast, the Dϕg residue adopts the more frequently observed fully extended conformation when the following residue adopts a semi-extended conformation (peptide a ). These findings are in agreement with the theoretical calculations on Ac-Dϕg-Aib-NHCH₃ and Ac-Aib-Dϕg-NHCH₃ also reported in this work. © 1998 European Peptide Society and John Wiley & Sons, Ltd.     

Chain length dependent transition of 310- to α-helix of Boc-(Ala-Aib)n-OMe

An apolar synthetic octapeptide, Boc-(Ala-Aib)₄-OMe, was crystallized in the triclinic space group P1 with cell dimensions a = 11.558 Å, b = 11.643 Å, c = 9.650 Å, α = 120.220°, β = 107.000°, γ = 90.430°, V = 1055.889 ų, Z = 1, C₃₄H₆₀O₁₁N₈·H₂O. The calculated crystal density was 1.217 g/cm³ and the absorption coefficient ? was 6.1. All the intrahelical hydrogen bonds are of the 3₁₀ type, but the torsion angles, ? and ψ, of Ala(5) and Ala(7) deviate from the standard values. The distortion of the 3₁₀-helix at the C-terminal half is due to accommodation of the bulky Boc group of an adjacent peptide in the nacking. A water molecule is held between the N-terminal of one peptide and the C-terminal of the other. The oxygen atom of water forms hydrogen bonds with N (1) -H and N (2) -H, which are not involved in the intrahelical hydrogen bonds. The hydrogen atoms of water also formed hydrogen bonds with carbonyl oxygens of the adjacent peptide molecule. On the other hand, ¹H-nmr analysis revealed that the octapeptide took an α-helical structure in a CD₃CN solution. The longer peptides, Boc-(Ala-Aib)₆-OMe and Boc-(Ala-Aib)₈-OMe, were also shown to take an α-helical structure in a CD₃CN solution. An α-helical conformation of the hexadecapeptide in the solid state was suggested by x-ray analysis of the crystalline structure. Thus, the critical length for transition from the 3₁₀- to α-helix of Boc-(Ala-Aib)_n-OMe is 8. © 1993 John Wiley & Sons, Inc.     

Theoretical conformational analysis of the opioid δ antagonist H-Tyr-Tic-Phe-OH and the μ agonist H-Tyr-D-Tic-Phe-NH2

A molecular mechanics study (grid search and energy minimization) of the highly δ receptor-selective δ opioid antagonist H-Tyr-Tic-Phe-OH (TIP; Tic: tetrahydroisoquinoline-3-car-boxylic acid) resulted in four low energy conformers with energies within 2 kcal/mol of that of the lowest energy structure. These four conformers contain trans peptide bonds only and represent compact structures showing various patterns of aromatic ring stacking. The centrally located Tic residue imposes several conformational constraints on the N-terminal dipeptide segment; however, the results of molecular dynamics simulations indicated that this tripeptide still shows some structural flexibility, particularly at the Phe³ residue. Analogous studies performed with the structurally related μ receptor-selective μ agonist H-Tyr-D -Tic-Phe-NH₂ resulted in low energy structures that were also compact but showed patterns of ring stacking different from those obtained with TIP. Superim-position of low energy conformers of TIP and H-Tyr-D -Tic-Phe-NH₂ revealed that the Phe³ residues of the L -Tic- and the D -Tic peptide were always located on opposite sides of the plane defined by the Tic residue, thus providing an explanation for the distinct activity profiles of the two compounds in structural terms. Attempts to demonstrate spatial overlap between the pharmacophoric moieties of low energy conformers of TIP and the nonpeptide δ antagonist naltrindole were made by superimposing either the Tyr¹ and Tic² aromatic rings and the N-terminal amino group or the Tyr¹ and Phe³ aromatic rings and the N-terminal amino group of the peptide with the corresponding aromatic rings and nitrogen atom in the alkaloid structure. In each case a low energy structure of TIP was found that showed good spatial overlap of all three specified pharmacophoric groups. These two conformers may represent candidate structures for the δ receptor-bound conformation of TIP. © 1994 John Wiley & Sons, Inc.     

Crystallographic characterization of the α,γ C12 helix in hybrid peptide sequences

The solid‐state conformations of two αγ hybrid peptides Boc‐[Aib‐γ⁴(R)Ile]₄‐OMe 1 and Boc‐[Aib‐γ⁴(R)Ile]₅‐OMe 2 are described. Peptides 1 and 2 adopt C₁₂‐helical conformations in crystals. The structure of octapeptide 1 is stabilized by six intramolecular 4 → 1 hydrogen bonds, forming 12 atom C₁₂ motifs. The structure of peptide 2 reveals the formation of eight successive C₁₂ hydrogen‐bonded turns. Average backbone dihedral angles for αγ C₁₂ helices are peptide 1 , Aib; φ (°) = ?57.2 ± 0.8, ψ (°) = ?44.5 ± 4.7; γ⁴(R)Ile; φ (°) = ?127.3 ± 7.3, θ₁ (°) = 58.5 ± 12.1, θ₂ (°) = 67.6 ± 10.1, ψ (°) = ?126.2 ± 16.1; peptide 2 , Aib; φ (°) = ?58.8 ± 5.1, ψ (°) = ?40.3 ± 5.5; ψ⁴(R)Ile; φ (°) = ?123.9 ± 2.7, θ₁ (°) = 53.3 θ 4.9, θ ₂ (°) = 61.2 ± 1.6, ψ (°) = ?121.8 ± 5.1. The tendency of γ⁴‐substituted residues to adopt gauche–gauche conformations about the C^α–C^β and C^β–C^γ bonds facilitates helical folding. The αγ C₁₂ helix is a backbone expanded analog of α peptide 3₁₀ helix. The hydrogen bond parameters for α peptide 3₁₀ and α‐helices are compared with those for αγ hybrid C₁₂ helix. Copyright © 2016 European Peptide Society and John Wiley & Sons.     

Helix-Forming tendencies of amino acids depend on their sequence contexts: Tripeptides AFG and FAG show incipient β-bulge formation in their crystal structures

Many of the theoretical methods used for predicting the occurrence of α-helices in peptides are based on the helical preferences of amino acid monomer residues. In order to check whether the helix-forming tendencies are based on helical preferences of monomers only or also on their sequence contexts, we synthesized permuted sequences of the tripeptides GAP, GAV, and GAL that formed crystalline helices with near α-helical conformation. The tripeptides AFG and FAG formed good crystals. The x-ray crystallographic studies of AFG and FAG showed that though they contain the same amino acids as GAF but in different sequences, they do not assume a helical conformation in the solid state. On the other hand, AFG and FAG, which contain the same amino acids but in a different sequence, exhibit nearly the same backbone torsion angles corresponding to an incipient formation of a β-bulge, and exhibit nearly identical unit cells and crystal structures. Based on these results, it appears that the helix-forming tendencies of amino acids depend on the sequence context in which it occurs in a polypeptide. The synthetic peptides AFG (L -Ala-L -Phe-Gly) and FAG (L -Phe-L -Ala-Gly), C₁₄H₁₉N₃O₄, crystallize in the orthorhombic space group P2₁2₁2₁, with a = 5. 232(1), b = 14. 622(2), c = 19. 157(3) Å, D_x = 1.329 g cm^?3, Z = 4, R = 0.041 for 549 reflections for AFG, and with a = 5. 488(2), b = 14.189 (1), c = 18.562(1) Å, D_x = 1.348 g cm^?3, Z = 4, R = 0.038 for 919 reflections for FAG. Unlike the other tripeptides GAF, GGV, GAL, and GAI, the crystals of AFG and FAG do not contain water molecule, and the molecules of AFG or FAG do not show the helical conformation. The torsion angles at the backbone of the peptide are ψ₁ = 144. 5(5)°; ?₂, ψ₂ = ?98.1(6)°, ?65.2(6)° ?₃, ψ₁₃, ψ₃₁ = 154.1(6)°, ?173.6(6)°, 6.9(8)° for AFG; and ψ₁ = 162.6(3)°; ?₂, ψ₂ = ?96.7(4)°, ?46.3(4)°; ?₃, ψ₁₃, ψ₃₁ = 150.1(3)°, ?168.7(3)°, 12.2(5)° for FAG. The conformation angles (? ψ) for residues 2 and 3 for both AFG and FAG show incipient formation of an β-bulge. © 1993 John Wiley & Sons, Inc.     

Δ53β hydroxysteroid dehydrogenase activity of the rat corpus luteum exhibits positive substrate-binding co-operativity: A continuous monitoring microdensitometric study with unfixed ovarian tissue sections

Δ⁵3β hydroxysteroid dehydrogenase activity transforms biologically inactive Δ⁵3β hydroxy steroids into the active Δ⁴3-keto products (e.g. pregnenolone to progesterone). Using a cytochemical procedure which allows for the continuous microdensitometric monitoring of an enzyme reaction as it proceeds and a well described cytochemical assay for Δ⁵3β HSD we have analysed the initial velocity rates (V_o) for dehydroepiandrosterone (DHEA) binding to this enzyme in regressing (i.e. 20α hydroxy steroid dehydrogenase positive) corpus luteum (CL) cells in unfixed tissue sections (5 μm) of the dioestrous and proestrous rat ovary. The results are mean ± S.E.M. The relationship between DHEA concentration (0 to 50 μM) and Δ⁵3β HSD activity in the dioestrous corpora lutea was sigmoidal and had an atypical 1/V_o versus 1/S plot, the x intercept being positive. Using a 1/V_o versus 1/S² plot the V_max was determined to be 1·0 ± 0·08 μmol min^?1 mg^?1 CL (n = 6). The Hill constant was 2·7 ± 0·02 (n = 6) suggesting a high degree of positive co-operativity for DHEA binding. The S concentration for half maximal activity was 17 ± 1 μmoles (n = 6). In the corpora lutea cells of the proestrous ovary, the V_max for DHEA transformation was unchanged (0·95 ± 0·04 μmol min^?1 mg^?1, n = 3) whilst the S_0·5 was significantly increased to 27 ± 0·1 (p < 0·01, n = 3). The Hill constant remained positive being 2·9 ± 0·2 (n = 3). NAD⁺ binding to 3β HSD in regressing corpora lutea of the proestrous ovary has been demonstrated previously to be hyperbolic and fit the classical Michaelis-Menten model.¹ Extending the analysis of NAD⁺ binding to the regressing corpus luteum of the dioestrous rat ovary revealed similar kinetic characteristics to that seen with the proestrous enzyme, the apparent V_max and K_m being 0·84 ± 0·04 μmol min^?1 mg^?1 CL (n = 3) and 27 ± 7 μmol 1^?1 (n = 3) respectively. The Hill constant was 1·1 ± 0·03 (n = 3), indicating no co-operativity of co-factor binding.     

A crystal structure with features of an antiparallel α-pleated sheet

A single-crystal x-ray diffraction analysis of Boc-L -Ala-D -aIle-L -Ile-OMe has been carried out. The analysis has shown (a) that the tripeptide molecules have in part an α-extended conformation, the torsion angles of the L -Ala and D -aIle residues being φ₁ = ?75.1° and ψ₁ = ?25.8° and φ₂ = 67.3° and ψ₂ = 44.1°, respectively, and (b) that the molecules are organized in rippled planes where they occur in relative antiparallel orientation linked together side by side by H bonds. This molecular organization of the tripeptide corresponds closely to that of an antiparallel α-pleated sheet, and likely constitutes the first example of a structure of this kind for which a characterization at the atomic level has been achieved. A molecular dynamics study has shown that the molecular conformation of the tripeptide in the crystalline state is determined primarily by intermolecular interactions. © 1994 John Wiley & Sons, Inc.     

X-Pro peptides: Solution and solid-state conformation of benzyloxycarbonyl-(Aib-Pro)2methyl ester,a type I β-turn

The synthesis of the tetrapeptide benzyloxycarbonyl(α-aminoisobutyryl-L -prolyl)₂-methyl ester (Z-(Aib-Pro)₂-OMe) and an analysis of its conformation in solution and the solid state are reported. Stepwise synthesis using dicyclohexylcarbodiimide leads to racemization at Pro(2). Evidence for the presence of diastereomeric tetrapeptides is obtained from 270-MHz¹H-nmr and 67.89-MHz ¹³C-nmr. The all-L tetrapeptide is obtained by fractional crystallization from ethyl acetate. The NH of Aib(3) is shown to be involved in an intramo-lecular hydrogen bond by variable-temperature ¹H-nmr and the solvent dependence of NH chemical shifts. The results are consistent with a β-turn conformation with Aib(1) and Pro(2) at the corners stabilized by a 4 → 1 hydrogen bond. The molecule crystallizes in the space group P2₁2₁2₁, with a = 8.839, b = 14.938, and c = 22.015 Å. The structure has been refined to an R value of 0.051. The peptide backbone is all-trans, and a 4 → 1 hydrogen bond, between the CO group of the urethane moiety and Aib(3) NH, is observed. Aib(1) and Pro(2) occupy the corner positions of a type I β-turn with ? = ?55.4°, Ψ = ?31.3° for Aib(1) and ? = ?71.6°, Ψ = ?38° for Pro(2). The tertiary amide unit linking Pro(2) and Aib(3) is significantly distorted from planarity (Δω = 14.3°).     

Characterization of the bioactive form of linear peptide antagonists at the δ-opioid receptor

The stereochemical requirements for δ-opioid receptor binding of a series of linear peptide antagonists with a novel conformationally restricted Phe analogue (Tic) as a second residue were examined by using a variety of computational chemistry methods. The δ-opioid receptor analogues with significant affinity, Tyr-Tic-NH₂ (TI-NH₂), Tyr-Tic-Phe-OH (TIP), Tyr-Tic-Phe-NH₂(TIP-NH₂), Tyr-Tic-Phe-Phe-OH (TIPP), Tyr-Tic-Phe-Phe-NH₂) (TIPP-NH₂), and the low affinity δ-opioid peptides Tyr-Pro-Phe-Pro-NH₂ (morphiceptin) and Tyr-Phe-Phe-Phe-NH₂ (TPPP-NH₂), were included in this study. The conformational profiles of these peptides were obtained by consecutive cycles of high and low temperature molecular dynamic simulations, coupled to molecular mechanical energy minimization carried out until no new conformational minima were obtained. Comparing the results for TPPP-NH₂ and TIPP-NH₂, the presence of the conformationally restricted Tic residue did not greatly reduce the number of unique low energy conformations, but did allow low energy conformers involving cis bonds between the first two residues. The conformational libraries of these peptides were examined for their ability to satisfy the three key ligand components for receptor recognition already identified by previous studies of high affinity cyclic (Tyr¹-D -Pen²-Gly³-Phe⁴-D -Pen⁵) enkephalin (DPDPE) type agonists: a protonated amine group, an aromatic ring, and a lipophilic moiety in a specific geometric arrangement. Two types of conformations common to the five high δ-opioid affinity L -Tic analogues were found that satisfied these requirements, one with a cis and the other with a trans peptide bond between the Tyr¹ and Tic² residues. Moreover, both the Tic² and Phe³ residues could mimic the hydrophobic interactions with the receptor of the Phe⁴ moiety in the cyclic DPDPE type agonists, consistent with the appreciable affinity of both di-and tripeptides. The low δ-opioid receptor affinity of morphiceptin can be understood as the result of conformational preferences that prevent the fulfillment of this pharmacophore for recognition. © 1996 John Wiley & Sons, Inc.     

Peptide design 310-helical conformation of a linear pentapeptide containing two dehydrophenylalanines,Boc-Gly-ΔZPhe-Leu-ΔZPhe-Ala-NHCH3

α, β-Dehydroamino acids are expected to provide conformational constraint to the peptide backbone. A pentapeptide containing two dehydrophenylalanines (Δ^ZPhe) separated by one L -amino acid has been synthesized and its solid state conformation determined. The pentapeptide, Boc-Gly-Δ^ZPhe-Leu-Δ^ZPhe-Ala-NHCH₃, crystallizes from aqueous methanol in the orthorhombic space group P2₁2₁2₁. There are four formula units, C₃₅H₄₆N₆O₇, in a unit cell of dimensions a = 10.155(3), b = 15.175(1), and c = 23.447(2) Å, at room temperature. The structure was solved by direct methods program, SIR88, and refined to a final R = 0.038 based on 3049 reflections with I > 2σ(I). All the peptide links are trans and the backbone conformation of the pentapeptide can be described as a 3₁₀-helix, with mean ?, ψ values of ?65.1° and ?22.8° (the value is averaged over the first four residues). There are four intramolecular 4 → 1 type hydrogen bonds characteristic of 3₁₀-type helices. In the crystal, the helices are held together by intermolecular N? H…?O?C head-to-tail and lateral hydrogen bonding between symmetry related molecules. This mode of packing is similar to the packing motifs observed so often in other oligopeptides that adopt a 3₁₀-helical structure. © 1993 John Wiley & Sons, Inc.     

Determination of β-glucosidase aggregating factor (BGAF) binding and polymerization regions on the maize β-glucosidase isozyme Glu1

β-Glucosidases (Glu1 and Glu2) in maize specifically interact with a lectin called β-glucosidase aggregating factor (BGAF). We have shown that the N-terminal (Glu⁵⁰–Val¹⁴⁵) and the C-terminal (Phe⁴⁶⁶–Ala⁵¹²) regions of maize Glu1 are involved in binding to BGAF. Sequence comparison between sorghum β-glucosidases (dhurrinases, which do not bind to BGAF) and maize β-glucosidases, and the 3D-structure of Glu1 suggested that the BGAF-binding site on Glu1 is much smaller than predicted previously. To define more precisely the BGAF-binding site, we constructed additional chimeric β-glucosidases. The results showed that a region spanning 11 amino acids (Ile⁷²–Thr⁸²) on Glu1 is essential and sufficient for BGAF binding, whereas the extreme N-terminal region Ser¹–Thr²⁹, together with C-terminal region Phe⁴⁶⁶–Ala⁵¹², affects the size of Glu1–BGAF complexes. The dissociation constants (K_d) of chimeric β-glucosidase–BGAF interactions also demonstrated that the extreme N-terminal and C-terminal regions are important but not essential for binding. To confirm the importance of Ile⁷²–Thr⁸² on Glu1 for BGAF binding, we constructed a chimeric sorghum β-glucosidase, Dhr2 (C-11, Dhr2 whose Val⁷²–Glu⁸² region was replaced with the Ile⁷²–Thr⁸² region of Glu1). C-11 binds to BGAF, indicating that the Ile⁷²–Thr⁸² region is indeed a major interaction site on Glu1 involved in BGAF binding.