Conformation of a water-soluble β-sheet model peptide. A circular dichroism and Fourier-transform infrared spectroscopie study of double D-amino acid replacements

Among peptide secondary structures β-sheet domains have been much less intensively studied than α-helical conformations, mainly because of the lack of well characterized model peptides. In the present paper the secondary structure of a water-soluble de novo peptide consisting of 26 amino acids (DPKGDPKGVTVTVTVTVTGKGDPKPD-NH₂) and the corresponding double D-amino acid replacement set have been studied by circular dichroism and Fourier-transform infrared spectroscopy. The model peptide was found to be unstructured in aqueous solution at peptide concentrations < 10^?3 mol/L but to adopt a predominantly β-sheet structure in the presence of 15 mM sodium dodecyl sulfate or at apolar/water interfaces. Although the peptide is composed of amino acids with low helical propensity, it formed a single-stranded helical structure in aqueous trifluoroethanol. The D-amino acid replacement set was synthesized in order to study the conformational stability of the model peptide selectively in distinct regions. The data show that both the α-helix present in 50% trifluoroethanol as well as the β-sheet domain formed in the presence of sodium dodecyl sulfate or at apolar/water interfaces, are located in the region between Val⁹ and Thr¹⁸. Pairwise substitution of adjacent amino acids by their corresponding D-amino acids provides a pronounced β-sheet disturbance. These findings demonstrate that double D-amino acid replacements may be used to locate β-sheet domains in peptides.     

Peptide models for β-turns.

The circular dichroism spectra of four β-turn model peptides, Z-Aib-Pro-Aib-Pro-OMe (1), Piv-Pro-Aib-NHMe (2), Piv-Pro-D-Ala-NHMe (3) and Piv-Pro-Val-NHMe (4) have been examined under a wide range of solvent conditions, using methanol, hexafluoroisopropanol and cyclohexane. Type I and Type II β-turns have been observed for peptides 1 and 2 respectively, in the solid state, while the Pro-D-Ala sequence adopts a Type II β-turn in a related peptide crystal structure. A class C spectrum is observed for 1 in various solvents, suggesting a variant of a Type I (III) structure. The Type II β-turn is characterized by a CD spectrum having two positive CD bands at ? 230 nm and ? 202 nm, a feature observed in Piv-Pro-D-Ala-NHMe in cyclohexane and methanol and for Piv-Pro-Aib-NHMe in methanol. Peptide 2 exhibits solvent dependent CD spectra, which may be rationalized by considering Type II, III and V reverse turn structures. Piv-Pro-Val-NHMe adopts non-β-turn structures in polar solvents, but exhibits a class B spectrum in cyclohexane suggesting a population of Type I β-turns.     

Conformation—activity correlations for chemotactic tripeptide analogs incorporating dialkyl residues with linear and cyclic alkyl sidechains at position 2

Five stereochemically constrained analogs of the chemotactic tripeptide incorporating l-aminocycloalkane-l-carboxylic acid (Ac_nc) and α, α-dialkylglycines (Deg, diethylglycine; Dpg, N, N-dipropylglycine and Dbg, N, N-dibutylglycine) at position 2 have been synthesized. NMR studies of peptides For-Met-Xxx-Phe-OMe (Xxx = Ac₇c. I: Ac₈c. II: Deg, III; Dpg, IV and Dbg, V; For, formyl) establish that peptides with cycloalkyl residues, I and II, adopt folded β-turn conformations in CDCl₃, and (CD³)₂SO. In contrast, analogs with linear alkyl sidechains, III-V, favour fully extended (C₅) conformations in solution. Peptides I-V exhibit high activity in inducing β-glucosaminidase release from rabbit neutrophils, with ED₅₀ values ranging from 1.4–8.0 × 10–¹¹. M. In human neutrophils the Dxg peptides III-V have ED₅₀ values ranging from 2.3 × 10^?8 to 5.9 × 10^?10 M, with the activity order being V>IV>III. While peptides I-IV are less active than the parent. For-Met-Leu-Phe-OH, in stimulating histamine release from human basophils, the Dbg peptide V is appreciably more potent, suggesting its potential utility as a probe for formyl peptide receptors. © Munksgaard 1996.     

Circular dichroism studies of helical oligopeptides: Can 310 and α-helical conformations be chiroptically distinguished?

Circular dichroism studies of seven helical oligopeptides containing α-aminoisobutyric acid (Aib) in methanol and trifluoroethanol (TFE) solutions are reported. Peptides ranging from 10 to 21 residues in length have been examined. In all cases distinct negative CD bands characteristic of helical peptides are obtained at 220 and 205 nm corresponding to the n-π* π-π* transitions, respectively. The ratio R = [θ]n-π*/[θ]π-π* is < 1.0 for all peptides studied. Using crystal structure and n.m.r. results for a 10 residue 310 helical peptide and literature values for an α-helical 11-residue peptide, it is shown that both helical conformations yield R values of ? 0.8 in alcoholic solvents. The CD data are considered in the light of ¹H n.m.r. studies on these oligopeptides. The results suggest that 310 and α-helical conformations cannot be distinguished by CD methods.     

Conformations of dehydrophenylalanine containing peptides Nuclear Overhauser effect study of two acyclic tetrapeptides

Two isomeric, acyclic tetrapeptides containing a Z-dehydrophenylalanine residue (Δ^z-Phe) at position 2 or 3, Boc-Leu-Ala-Δ^z-Phe-Leu-OMe (1) and Boc-Leu-Δ^z-Phe-Ala-Leu-OMe (2), have been synthesized and their solution conformations investigated by 270MHz ¹H n.m.r. spectroscopy. In peptide 1 the Leu(4) NH group appears to be partially shielded from solvent, while in peptide 2 both Ala(3) and Leu(4) NH groups show limited solvent accessibility. Extensive difference nuclear Overhauser effect (n.O.e.) studies establish the occurrence of several diagnostic inter-residue n.O.e.s (Cα_jH ? N_i+1H and N_iH ? N_i+1H) between backbone protons. The simultaneous observation of “mutually exclusive” n.O.e.s suggests the presence of multiple solution conformations for both peptides. In peptide 1 the n.O.e. data are consistent with a dynamic equilibrium between an -Ala-Δ^z-Phe- Type II β-turn structure and a second species with Δ^z-Phe adopting a partially extended conformation with Ψ values of ± 100° to ± 150°. In peptide 2 the results are compatible with an equilibrium between a highly folded consecutive β-turn structure for the -Leu-Δ^z-Phe-Ala- segment and an almost completely extended conformation.     

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.     

Peptides from chiral Cα,α-disubstituted glycines

The molecular and crystal structures of one derivative and three model peptides (to the pentapeptide level) of the chiral C^α,α-disubstituted glycine Cα-methyl, Cα-isopropylglycine [(αMe)Val] have been determined by X-ray diffraction. The derivative is mClAc-l -(α Me)Val-OH, and the peptides are Z-l -(αMe)Val-(l -Ala)₂-OMe monohydrate, Z-Aib-L-(αMe)Val-(Aib)₂-OtBu, and Ac-(Aib)₂-l -(αMe)Val-(Aib)₂OtBu acetonitrile solvate. The tripeptide adopts a type-I β-turn conformation stabilized by a 1 ← 4N-H . O=C intramolecular H-bond. The tetra- and pentapeptides are folded in regular right-handed 3₁₀-helices. All four L-(αMe)Val residues prefer φ, Ψ angles in the right-handed helical region of the conformational map. The results indicate that: (i) the (αMe)Val residue is a strong type-I/III β-turn and helix former, and (ii) the relationship between (αMe)Val chirality and helix screw sense is the same as that of Cα-monosubstituted protein amino-acids. The implications for the use of the (αMe)Val residue in designing conformationally constrained analogues of bioactive peptides are briefly discussed.     

Conformations of peptides containing 1-aminocyclohexanecarboxylic acid (Acc6). Crystal structures of two model peptides

The crystal structures of two peptides containing 1-aminocyclohexanecarboxylic acid (Acc⁶) are described. Boc-Aib-Acc⁶-NHMe · H₂O adopts a β-turn conformation in the solid state, stabilized by an intramolecular 4 → 1 hydrogen bond between the Boc CO and methylamide NH groups. The backbone conformational angles (φ_Aib = – 50.3°, ψ_Aib = – 45.8°; φ_Acc6 = – 68.4°, ψ_Acc6 = – 15°) lie in between the values expected for ideal Type I or III β-turns. In Boc-Aib-Acc⁶-OMe, the Aib residue adopts a partially extended conformation (φ_Aib = – 62.2°, ψ_Aib = 143°) while the Acc⁶residue maintains a helical conformation (φ_Acc6 = 48°, ψ_Acc⁶= 42.6°). ¹H n.m.r. studies in CDCl₃ and (CD₃)₂SO suggest that Boc-Aib-Acc⁶-NHMe maintains the β-turn conformation in solution.     

Circular dichroic and immunological properties of human choriogonadotropin-β carboxyl terminal peptides

Circular dichroic spectra have been obtained in aqueous solution and in trifluoroethanol for several synthetic (non-glycosylated) human choriogonadotropin carboxyl terminal peptides of the β-subunit ranging in size from 10 residues to 40 residues. There was no evidence for formation of α-helicity or β-structure, but the spectra in 90% (v/v) trifluoroethanol were consistent with the occurrence of β-turns. The Chou-Fasman predictive rules also suggest a high probability of β-turns in these peptides which could result in the occurrence of repeating kinks. Disulfide-linked dimers were also investigated by circular dichroism, and there was evidence of stabilization of particular skewness of the disulfide dihedral angle depending upon the location of the disulfide bond. The single phenylalanyl residue at position 115 in the β-subunit also contributed to the circular dichroic spectra above 250 nm. Antibodies raised to a peptide consisting of residues 111–145 have been shown to contain two immunological determinants, but the sum of antibodies raised to separate determinant sequences do not equal those raised to the full length peptide. These data could reflect the existence of a conformation-related determinant on the 111–145 peptide or stearic hindrance of immunoglobulin binding of two antibodies to the same peptide.     

New tools for the control of peptide conformation: the helicogenic Cα‐methyl,Cα‐cyclohexylglycine*

Abstract: The novel C^α‐tetrasubstituted α‐amino acid C^α‐methyl, C^α‐cyclohexylglycine was prepared by hydrogenation of its C^α‐methyl, C^α‐phenylglycine precursor. Terminally protected homodi‐, homotri‐, and homotetrapeptides from C^α‐methyl, C^α‐cyclohexylglycine and co‐oligopeptides to the pentamer level in combination with Gly or α‐aminoisobutyric acid residues were prepared by solution methods and fully characterized. The results of a conformational analysis, performed by use of Fourier transform infrared (FT‐IR) spectrophotomet absorption, ¹H NMR, and X‐ray diffraction techniques, support the contention that this C^α‐methylated, C^β‐trisubstituted aliphatic α‐amino acid is an effective β‐turn and 3₁₀‐helix inducer in tri‐ and longer peptides as its C^α‐methyl valine parent compound, but partially divergent from the corresponding aromatic C^α‐methyl, C^α‐diphenylmethylglycine residue, known to promote folded and fully extended structures to a significant extent in these oligomers.     

Glycosylation of synthetic peptides breaks helices

Two proposed glycosylation sites are located within T cell epitopes of rabies virus glycoprotein, namely VVEDEGCTNLSGF (VF13; amino acids 29-41) and GKAYTIFNKTLM (GM12; amino acids 312-323). To explore the effects on peptide conformation due to post-translational modifications, we synthesized glycosylated and phosphorylated versions of the two peptides and compared their structures with the native peptide using CD and FT-IR spectroscopy. After the modifications, i.e., glycosylation on Asn with one or two N-acetyl-glucosamine or glucose residues or phosphorylation on Ser, the low to medium degree of helicity of the unmodified peptides disappears as indicated by CD measurements in water-trifluoroethanol mixtures. Incorporation of one sugar moiety into either peptide resulted with a high probability in a type I (III) β-turn formation with almost identical spectra for the different peptides. Elongation of the carbohydrate in GM12 only slightly enhanced this effect. In contrast, phosphorylation of VF13 caused distorted conformation of the peptide backbone. This novel and direct demonstration of a change in secondary structure by glycosylation (or phosphorylation) might be an important element in determining peptide antigen structure and function.     

H N.M.R. STUDIES OF PROTECTED α-AMINOISOBUTYRIC ACID CONTAINING PEPTIDES

The benzylic methylene protons in a large number of benzyloxycarbonyl α-aminoisobutyric acid (Z-Aib) containing peptides, show chemical shift nonequivalence. The magnitude of the geminal nonequivalence is correlated with the involvement of the urethane carbonyl group, in an intramolecular hydrogen bond. Studies of the model compounds Z-Aib-Aib-Ala-NHMe, and Z-Aib-Aib-Aib-Pro-OMe clearly establish the presence of intramolecular hydrogen bonds, involving the urethane CO group. In both compounds marked anisochrony of the benzylic methylene protons is demonstrated. In Z-Aib-Aib-Pro-OMe, where a 4 → 1 hydrogen bonded β-turn is not possible, the benzylic -CH₂- protons appear as a singlet in CDCl₃ and have a very small chemical shift difference in (CD₃)₂SO. The observation of such nonequivalence is of value in establishing whether the amino terminal Aib-Pro β-turn is retained in large peptide fragments of alamethicin.     

The twists and turns of β-peptides

Abstract: Recently, it has been discovered that peptides composed of β-amino acids are capable of adopting novel secondary structures demonstrating that peptides composed of α-amino acids are not unique in their ability to fold into well-defined structures. Cyclic as well as acyclic peptides composed of β-amino acid residues adopt turn, helical, and sheet-like conformations. Here, we discuss the synthesis and conformational preferences of individual, substituted β-amino acids as well as the structures that peptides composed of these residues, β-peptides, may adopt.     

Peptides from chiral Cα,α-disubstituted glycines Synthesis and characterization,conformational energy computations and solution conformational analysis of Cα-methyl,Cα-isopropylglycine [(αMe)Val] derivatives and model peptides*

Conformational energy computations on Ac-l -(αMe)Val-NHMe indicate that turns and right-handed helical structures are particularly stable conformations for this chiral Cα-methyl, Cα-alkylglycyl residue. We have synthesized and characterized a variety of l -(αMe)Val derivatives and peptides (to the pentamer level). The results of the solution conformational analysis, performed using infrared absorption, ¹H nuclear magnetic resonance, and circular dichroism, are in general agreement with those obtained from the theoretical investigation, in the sense that the l -(αMe)Val residue turns out to be a strong β-turn and right-handed helix former. A comparison is also made with the conclusions extracted from published work on peptides rich in other Cα-methyl, Cα-alkylglycyl residues.     

Stereochemical analysis of the antigenic tip of the V3 loop peptide of HIV-1 gp120

A novel multiple turn conformation has been observed for a segment GPGRAFY in the crystal structure of a complex of HIV-1 gp120 V3 loop peptide with the Fab fragment of a neutralizing antibody [Ghiara et al. (1994) Science 264 , 82–85]. A structural motif has been defined for the peptide segment, employing idealized backbone conformations characterized by ranges of virtual C^α torsion angles and bond angles. A search of 122 high-resolution protein crystal structures has permitted identification of 24 examples of similar structural motifs. Two major conformational families have been identified, which differ primarily in the conformation at residue 3. The observed conformation at residue 3 in family 1 is left-handed helical (α_L) and that in family 2 is right-handed helical (α_R). Of the 10 examples in family 1, 9 examples have Gly residues at position 3. Of the 12 examples in family 2, 7 examples have Asn/Asp at position 3. Computer modeling of the V3 loop tip sequence using the two backbone conformational families as starting points leads to minimum-energy conformations in which antigenically important side-chains occupy similar spatial arrangements. This stereo-chemical analysis of the V3 loop tip sequence suggests a rational basis for the design of synthetic analog peptides for use as viral antagonists or synthetic antigens. © Munksgaard 1995.     

Molecular design of peptides

The peptide N-Boc-l -Phe-dehydro-Abu-NH-CH₃ was synthesized by the usual workup procedure. The crystals grown from methanol at 4°C belong to the space group P2₁2₁2₁ with a= 7.589(2), b= 13.690(4), c= 21.897(6) Å, Z= 4 and d_c= 1.149(5) g cm^?3 for C₁₉H₂₉N₃O₅·CH₃OH. The peptide crystals were highly sensitive to radiation. The final agreement factor R was 0.055 for 1109 observed reflections (I > 2σ) with data extending to a 2θ value of 103°. The methanol oxygen atom is split into two occupancies. Both sites are involved in identical hydrogen bonding. As a result of substitution of a dehydro-Abu residue at the (i+ 2) position the peptide adopts an ideal β-turn II′ conformation with torsion angles of corner residues as φ₁=63(1)°, ψ₁= - 127(1)°, φ₂= -66(1)° and ψ₂= - 10(1)°, and an intramolecular hydrogen bond N—H ? O of length 3.01(1) Å. This shows that the conformational constraints produced by dehydro-Abu are similar in nature to but different in magnitude than those produced by dehydro-Phe and dehydro-Leu. The methanol–peptide interactions show characteristic features of multiple hydrogen-bond formations involving polar sites of participating peptide and methanol molecules. The packing of the molecules in the unit cell is stabilized by interactions through methanol molecules with the help of several hydrogen bonds.     

Conformational properties of hybrid peptides containing α‐ and ω‐amino acids*

Abstract: This review briefly surveys the conformational properties of guest ω‐amino acid residues when incorporated into host α‐peptide sequences. The results presented focus primarily on the use of β‐ and γ‐residues in αω sequences. The insertion of additional methylene groups into peptide backbones enhances the range of accessible conformations, introducing additional torsional variables. A nomenclature system, which permits ready comparisons between α‐peptides and hybrid sequences, is defined. Crystal structure determination of hybrid peptides, which adopt helical and β‐hairpin conformations permits the characterization of backbone conformational parameters for β‐ and γ‐residues inserted into regular α‐polypeptide structures. Substituted β‐ and γ‐residues are more limited in the range of accessible conformation than their unsubstituted counterparts. The achiral β,β‐disubstituted γ‐amino acid, gabapentin, is an example of a stereochemically constrained residue in which the torsion angles about the C^β–C^γ (θ₁) and C^α–C^β (θ₂) bonds are restricted to the gauche conformation. Hybrid sequences permit the design of novel hydrogen bonded rings in peptide structures.     

Amphipathic control of the 310‐/α‐helix equilibrium in synthetic peptides

Abstract: A series of short, amphipathic peptides incorporating 80% C^α,C^α‐disubstituted glycines has been prepared to investigate amphipathicity as a helix‐stabilizing effect. The peptides were designed to adopt 3₁₀‐ or α‐helices based on amphipathic design of the primary sequence. Characterization by circular dichroism spectroscopy in various media (1 : 1 acetonitrile/water; 9 : 1 acetonitrile/water; 9 : 1 acetonitrile/TFE; 25 mm SDS micelles in water) indicates that the peptides selectively adopt their designed conformation in micellar environments. We speculate that steric effects from ith and ith + 3 residues interactions may destabilize the 3₁₀‐helix in peptides containing amino acids with large side‐chains, as with 1‐aminocyclohexane‐1‐carboxylic acid (Ac₆c). This problem may be overcome by alternating large and small amino acids in the ith and ith + 3 residues, which are staggered in the 3₁₀‐helix.     

Stereochemistry of peptides containing 1-aminocycloheptane-1-carboxylic acid (Ac7c)

The crystal structures of four peptides incorporating l-aminocycloheptane-l-carboxylic acid (Ac₇c) are described. Boc-Aib-Ac₇c-NHMe and Boc-Pro-Ac₇c-Ala-OMe adopt β-turn conformations stabilized by an intramolecular 4 × 1 hydrogen bond, the former folding into a type-I/III β-turn and the latter into a type-II β-turn. In the dipeptide esters, Boc-Aib-Ac₇c-OMe and Boc-Pro-Ac₇c-OMe, the Ac₇c and Aib residues adopt helical conformations, while the Pro residue remains semi-extended in both the molecules of Boc-Pro-Ac₇c-OMe found in the asymmetric unit. The cycloheptane ring of Ac₇c residues adopts a twist-chair conformation in all the peptides studied. ¹H-NMR studies in CDCl₃ and (CD₃)₂SO and IR studies in CDCl₃, suggest that Boc-Aib-Ac₇c-NHMe and Boc-Pro-Ac₇c-Ala-OMe maintain the β-turn conformations in solution.     

Conformational investigation of α, β-dehydropeptides

The crystal structure of Ac-Pro-ΔVal-NHCH₃ was examined to determine the influence of the α,β-dehydrovaline residue on the nature of peptide conformation. The peptide crystallizes from methanol-diethyl ether solution at 4° in needle-shaped form in orthorhombic space group P2₁2₁2₁ with a= 11.384(2) Å, b = 13.277(2) Å, c = 9.942(1) Å. V = 1502.7(4) ų Z = 4, D_m= 1.17 g cm^?3 and D_c=1.18 g cm^?3 The structure was solved by direct methods using SHELXS-86 and refined to an R value of 0.057 for 1922 observed reflections. The peptide is found to adopt a β-bend between the type I and the type III conformation with φ₁=?68.3(4)°, ψ₁=? 20.1(4)°, φ₂=?73.5(4)°= and Ψ₂=?14.1(4)°=. An intramolecular hydrogen bond between the carbonyl oxygen of ith residue and the NH of (i+ 3)th residue stabilizes the β-bend. An additional intermolecular N.,.O hydrogen bond joins molecules into infinite chains. In the literature described crystal structures of peptides having a single α,β-dehydroamino acid residue in the (i+ 2) position and forming a β-bend reveal a type II conformation.