Conformational characteristics of asparaginyl residues in proteins

Backbone conformations at 1064 asparaginyl residues in 123 non-homologous, high-resolution X-ray structures of proteins were analysed. Asn adopts conformations in left-handed α-helical region and other partially allowed regions in the Ramachandran map more readily than any other non-glycyl residue. Asn conformational clusters in the (φ,ψ) regions of left-handed α-helix, right-handed 7ALPHA;-helix and extended (β) strands were investigated in detail for their occurrence in various secondary structures, especially in β-turn regions. Preferences were observed for Asn conformations in different positions in various β-turn types, including the first and fourth positions of the turn. Asparaginyl residues with extended conformations are found to occur frequently in irregular regions, although they are expected to occur predominantly in extended strands or in the third position of type II β-turns. Asn conformations at the N-cap positions of helices strongly prefer extended conformation than α₁, which seems to be characteristic of non-glycyl residues at that position. In the linkers connecting two extended strands and those connecting an α-helix and an extended strand, Asn with α¹ or α_R conformation is more favoured than Asn with the β-conformation. Analysis of Asn-Asn doublets and Asn-X-Asn triplets permitted identification of conformational families in such sequences. Results of this investigation provide useful hints in modelling Asn-rich regions in proteins such as malaria parasite coat protein.     

Design of peptides with αβ-dehydro residues: Synthesis,crystal structure and molecular conformation of N-Boc-L-Ile-ΔPhe-L-Trp-OCH3

The dehydro-peptide Boc-L-Ile-ΔPhe-L-Trp-OCH₃ was synthesized by the azlactone method in the solution phase. The peptide was crystallized from methanol in an orthorhombic space group P2₁2₁2₁ with a = 10.777(2), b= 11.224(2), c= 26.627(10) Å. The structure was determined by direct methods and refined to an R value of 0.069 for 3093 observed reflections [l≥ 2σ(l)].The peptide failed to adopt a folded conformation with backbone torsion angles: φ₁, = 90.8(8)°, ψ₁= -151.6(6)°, φ₂= 89.0(8)°, ψ₂= 15.9(9)°, φ₃= 165.7(7)°, ψ^T₃= -166.0(7)°. A general rule derived from earlier studies indicates that a three-peptide unit sequence with a ΔPhe at the (i+ 2) position adopts a β-turn II conformation. Because the branched β-carbon residues such as valine and isoleucine have strong conformational preferences, they combine with the ΔPhe residue differently to generate a unique set of conformations in such peptides. The presence of β-branched residues simultaneously at both (i+ 1) and (i+ 3) positions induces unfolded conformations in tetrapeptides, but a β-branched residue substituted only at (i+ 3) positron can not prevent the formation of a folded β-turn II conformation. On the other hand, the present structure shows that a β-branched residue substituted at the (i+ 1) position prevents the formation of a β-turn II conformation. These observations indicate that a β-branched residue at the (i+ 1) position prevents a folded conformation whereas it cannot generate the same degree of effect from the (i+ 3) position. This may be because of the trans disposition of the planar ΔPhe side-chain with respect to the C=O group in the residue. The molecules are packed in an anti-parallel manner to generate N₂-H₂…O₂ (-x,y-1/2, -z+ 3/2) and N^ε1₃-H^ε1₃…O₁(-x,y -1/2, -z+ 3/2) hydrogen bonds.     

Solution structure of a biologically active cyclic LDV peptide analogue containing a type II′β-turn mimetic

The solution structure of cyclo-[Gly-Leu-Asp-Val-BTD] (BTD=β-turn dipeptide) has been determined by two-dimensional ¹H-NMR (nuclear magnetic resonance) spectroscopy and systematic conformational searching combined with molecular dynamics studies. The structure contains two hydrogen bonds between the Gly and Val residues, and a type I β-turn with Leu and Asp at the (i+ 1) and (i+ 2) positions of the turn. The cyclic compound shows activity in a scintillation proximity assay (SPA) for the inhibition of the interaction between the integrin α₄β₁ and vascular cell adhesion molecule-1 (VCAM-1). The structure-activity relationship of the LDV sequence is discussed. © Munksgaard 1996.     

Determination of structural elements related to the biological activities of a potent decapeptide agonist of human C5a anaphylatoxin

Abstract: The structural features related to the biologic activities of a potent, response-selective decapeptide agonist of human C5a, YSFKPMPLaR (C5a_65–74, Y65, F67, P69, P71, d -Ala73), were identified by NMR analysis in H₂O, DMSO and TFE. This investigation showed that the KPM residues in H₂O and the SFKPM residues in DMSO exhibited an extended backbone conformation, whereas a twisted conformation was found in this region in TFE. In H₂O, the C-terminal region (PLaR) adopted a distorted type II β-turn or a type II/V β-turn. In the type II/V β-turn, Leu72 exhibited a conformation typical of a type II β-turn, whereas d -Ala73 exhibited a conformation characteristic of a type V β-turn. Furthermore, a γ-turn involving residues LaR overlapped with the type II/V β-turn. In DMSO, the C-terminal region had the analogous turn-like motif (type II/V β-turn overlapping with γ-turn) found in H₂O. In TFE, no β-turn motifs were formed by the PLaR residues. These turn-like motifs in the C-terminal region of the peptide in both H₂O and DMSO were in agreement with the biologically important conformations predicted earlier by a structure–function analysis of a related panel of decapeptide analogs. The motifs determined by the NMR analysis of YSFKPMPLaR in H₂O and DMSO may represent structural elements important for C5a agonist activity and thus can be used to design the next generation of C5a agonist, partial agonist and antagonist analogs.     

Influence of serine in position i on conformation and dynamics of reverse turns

NMR spectroscopy has been employed for the conformational analysis of the cyclic hexapeptide cycle(-d -Pro¹-Ala²-Ser³(Bzl)-Trp⁴-Orn⁵(Z)-Tyr⁶-) with and without protecting groups on Ser³ and Orn⁵. This peptide sequence was derived from the active loop sequence of the α-amylase inhibitor Tendamistat (HOE 467). The aim was to investigate the role of serine in position i of a standard β-turn on the conformation and stabilization of this turn. Based on distance and torsion constraints from 2D NMR spectroscopic measurements in DMSO-d₆ solution, structure refinement was accomplished by restrained molecular dynamics (MD) simulations in vacuo and in DMSO. The analysis of both structures in solution reveals a considerable effect of the unprotected serine sidechain on the adjacent β-turn conformation. While in the protected peptide with Ser³(Bzl) a βII-turn is observed between Trp⁴ and Orn⁵, the deprotected compound reveals a βI-turn in this region. The βI-turn is stabilized by a backbone-sidechain hydrogen bond from Orn⁵N_αH to Ser³O_γ. Comparisons with other NMR-derived solution structures of cyclic model peptides and in some protein structures from literature reveal a general structural motif in the stabilization of βI-turns by serine in the i position through backbone-sidechain interactions. © Munksgaard 1995.     

NMR analysis of a potent decapeptide agonist of human C5a anaphylatoxin

A NMR investigation in H₂0, TFE and DMSO of a conformationally constrained, potent decapeptide agonist of human C5a, YSFKDMPLaR (C5a₆₅-₇₄, Y65, F67, P71, d -Ala73) showed that its N-terminal region (YSFKD) exhibited an extended backbone conformation in H₂O and a more twisted conformation in both TFE/H₂O (30:70, v/v; referred to as TFE) and DMSO. The C-terminal region (MPLaR) of the peptide adopted compact, turn-like structures. In H₂O, the C-terminal region adopted a type II β-turn or a distorted type V/II β-turn involving residues PLaR. In the distorted type V/II β-turn, Leu72 exhibited a conformation typical of a type V β-turn, whereas D -Ala73 exhibited a conformation typical of a type II β-turn. The distorted type V/II β-turn overlapped with an inverse γ-turn involving residues MPL. In DMSO, the C-terminal region had the analogous inverse y-turn and the V/II γ-turn found in H₂O. In many of the DMSO structures, two inverse γ-turns in the MPL and PLa positions formed a double-inverse γ-turn. None of the turns observed in H₂O were present in TFE. However, in TFE, the PLa residues formed an inverse γ-turn. Overall, the turn-like structural motifs in the C-terminal region of the peptide in both H₂O and DMSO (but not in TFE) agreed with the biologically important conformations obtained earlier by the structure-function analysis of a panel of C5a agonist peptides. These motifs may represent key structural elements important for C5a agonist activity and may be used to design the next generation of C5a agonist and antagonist analogues. © Munksgaard 1998.     

Conformational analysis of homochiral and heterochiral diprolines as β-turn-forming peptidomimetics: unsubstituted and substituted models

The effect of replacing one of the proline residues in either unsubstituted homochiral or heterochiral diproline segments with either a 2- or a 3-substituted prolyl residue on the allowed conformation of the diproline template has been examined. In heterochiral (l-d ) diprolines, placement of a 2-methyl-d -proline residue in the i+ 2 position and placement of either a cis- or trans-3-methyl-l -proline residue in the i+ 1 position results in substituted diproline peptides that adopt the same type II β-turn conformation as that identified experimentally for the unsubstituted diproline peptides. In contrast, placement of a cis-3-methyl-d -proline residue in the i+ 1 position of a homochiral (d-d ) diproline peptide seems to promote a different conformation than that seen in the unsubstituted case, whereas the trans-3-methyl-d -proline residue seems to provide a stabilizing influence for the predicted type VI'β-turn. The demonstrated ability of certain substituted diproline templates to adopt predictable conformations coupled with the development of asymmetric synthetic routes to both 2- and 3-substituted prolyl residues capable of mimicking a variety of side chains should make these templates useful tools in designing specific turn mimics of biologically active molecules.     

Conformation and structure of linear peptides with regularly alternating l- and d-residues: structure of the blocked hexapeptide tert-butyloxycarbonyl-(d-alloisoleucyl-l-isoleucyl)3 methyl ester monohydrate

Peptides with a regular sequence of enantiomeric residues (l and d ) along the chain have received considerable attention because of their accessibility to unique conformations and because they are model compounds for the naturally occurring peptide gramicidin A, which shows monovalent cation selective transmembrane transport. The solid-state structure of the linear hexapeptide t-Boc-(d -aIle-l -Ile)₃-OMe has been determined by X-ray diffraction techniques and refined to a final R factor of 0.068. The molecule shows a bent U-shaped conformation stabilized by three intramolecular H-bonds of the N—H?O=C type: a type II β-bend (4 → 1 bend or C₁₀ ring structure) with l -Ile² and d -aIle³ at positions 2 and 3 of the bend, an α-turn (5 → 1 bend or C₁₃ ring structure) and a 1 → 5 bend or C₁₇ ring structure. The first two 10-membered and 13-membered bends are enclosed in the latter 17-membered hydrogen-bonded ring structure. This structural motif is common to hepta- and octa-peptide cyclic molecules, showing that ring closure is not required to achieve a particular topology in the molecular design of specific bended conformations. © Munksgaard 1995.     

Peptides related to the active fragment of “proline rich polypeptide”, an immunoregulatory protein of the ovine colostrums

The preferred solution conformation of the PRP-hexapeptide (Tyr-Val-Pro-Leu-Phe-Pro) and of some of its structural analogues was investigated by NMR- spectroscopy, spectrofluorimetry and computer simulation technic. It was found that the preferred conformation is characterized by cis′-conformation of Pro³ and the γ-turn on the Leu⁴-residue: for Val² and Phe⁵ a β-structure seems to be privileged. In such a conformation Val² and Leu⁴ residues occupy exactly the same positions in space as residues i and i+ 3 in an α-helix. It suggests that the PRP-hexapeptide can interact with receptor protein inducing or stabilizing its helical conformation by “knobs into holes” packing.     

Structural redesign and stabilization of the overlapping tandem β-turns of RNA polymerase II

Peptides representing single repeat units of the carboxy-terminal domain (CTD) of RNA polymerase II (Tyr-Ser-Pro-Thr-Ser-Pro-Ser-Tyr-NH₂, 1) contain overlapping Ser-Pro-Xaa-Xaa β-turn forming sites which permit their overall structure to closely resemble members of the quinoxaline class of antitumor DNA bisintercalators. We have modified this native sequence at the i+2 positions of each β-turn unit by substituting Gly or D-Ala in an attempt to preorganize this structure in aqueous solution. CD and NMR spectroscopic investigations confirmed the presence of type II β-turns within each of the substituted peptides in contrast to the native sequence which contains a relatively low population of turn structure. In addition, an examination of singly substituted peptides suggests that an increase in the population of β-turn structure within the amino-terminal Ser-Pro-Xaa-Xaa site also increased the formation of β-turn structure in the carboxy-terminal (unmodified) Ser-Pro-Xaa-Xaa site; in comparison, substitution in the carboxy-terminal site did not influence structure in the remaining portion of the peptide. Overall, these results suggest that the structures formed could provide unique. preorganized linkers for the construction of novel DNA-interactive bisintercalators. © Munksgaard 1996.     

The evaluation of type I and type II β-turn mixtures

Circular dichroism (CD) and¹ H-{¹H}NOE spectra were obtained for Piv-Pro-Ser-NHCH₃(1),[Piv-(CH₃)₃-C-CO], Boc-Pro-Ser-NHCH₃ (2) and Boc-Val-Ser-NHCH₃ (3), to determine the solution conformation of these p-turn models. In the crystal, 1 and 3 adopt an ideal type I β-turn, while 2 is characterized by a semifolded backbone geometry incorporating a cis Boc-Pro tert-amide bond. The predominance of a β-turn conformation in solution was suggested for models 1-3 on the basis of ¹H-{¹H}NOE data. In a nonpolar solvent the prevailing trans rotamer form (>80%) of 2 has a β-turn conformation according to heteronuclear NOE measurement. Positive ¹H-{¹H} NOEs were detected between the H^α(Pro)/NH(Ser), Hα(Ser)/NH(Ser) and NH(NHCH₃3)/HN(Ser) protons in the trans Boc-Pro rotamer form of 2 at -20° in CDCl₃. Similar positive homonuclear NOE enhancements were also observed on the appropriate proton signals in other models, such as Boc-Val-Ser-NHCH₃ (3). Boc-Val-D-Ser-NHCH₃ (4) and Boc-Pro-D-Ser-NHCH₃ (5), in various solvents. The ¹H- {¹H)NOE experiments carried out in CD₃CN clearly showed that besides the type I (or III) β-turn structure, one of the main conformations of models 1-5 is close to the type II β-turn backbone geometry in a nonpolar solvent. Unexpectedly, the conformational mixture of models 1-3 were characterized by class C (helix-like) CD spectra, although class C spectra are generally only correlated with the type I β-turn conformation. These acyclic models are the first carefully investigated examples of -L-L- triamide systems, containing a significant amount of a type II β-turn, as well as the type I p-turn and, however, yielding a class C circular dichroism spectra. The CD spectra recorded for 3 and 4 in acetonitrile were ‘calibrated’ using the ¹H-{¹H}NOE data. Such a “calibration”, as well as the semi-quantitative CD and NMR comprehensive analyses, demonstrated that class C, class B, as well as class C’ CD spectra may be obtained from the linear combination of the same two-component spectra, with different conformational weights. Therefore, it is suggested that the extraction of the conformational components of such models, simply on the basis of their CD spectra, must be made with caution.     

Evidence for a C-terminal turn in PBAN An NMR and distance geometry study

The solution conformation of the pheromone biosynthesis activating neuropeptide (PBAN) of the moth Helicoverpa zea has been determined using homonuclear two-dimensional nuclear magnetic resonance techniques and distance geometry-restrained energy minimization. The insect peptide hormone showed a random distribution of conformers in aqueous solution, whereas in a less polar medium of trifluoroethanol and water, a reordering process was observed. In particular, the C-terminal region (Phe-Ser-Pro-Arg-Leu-NH₂) adopts a type I′β-turn conformation, residues 20-27 are in a helix conformation, and residues 1-19 exhibit a high degree of flexibility. Direct observation of the C-terminal β-turn configuration of PBAN is consistent with a previous report that showed a rigid, cyclic analog of the C-terminal pentapeptide of PBAN retained pheromonotropic activity [Nachman, R.J., Kuniyoshi, H., Roberts, V.A., Holman, G.M. & Suzuki, A. (1993). Biochem. Biophys. Res. Commun. 661-666], Furthermore, our results show no interaction between the C-terminal turn and the rest of the polypeptide chain, thus providing further evidence that the C-terminal turn is indeed the important conformation recognized by the PBAN receptor. © Munksgaard 1996.     

Role of azaamino acid residue in β‐turn formation and stability in designed peptide

Abstract: The structural perturbation induced by C^αH→N^α exchange in azaamino acid‐containing peptides was predicted by ab initio calculation of the 6‐31G* and 3‐21G* levels. The global energy‐minimum conformations for model compounds, For‐azaXaa‐NH₂ (Xaa = Gly, Ala, Leu) appeared to be the β‐turn motif with a dihedral angle of φ = ± 90°, ψ = 0°. This suggests that incorporation of the azaXaa residue into the i + 2 position of designed peptides could stabilize the β‐turn structure. The model azaLeu‐containing peptide, Boc‐Phe‐azaLeu‐Ala‐OMe, which is predicted to adopt a β‐turn conformation was designed and synthesized in order to experimentally elucidate the role of the azaamino acid residue. Its structural preference in organic solvents was investigated using ¹H NMR, molecular modelling and IR spectroscopy. The temperature coefficients of amide protons, the characteristic NOE patterns, the restrained molecular dynamics simulation and IR spectroscopy defined the dihedral angles [ (φ_i+1, ψ_i+1) (φ_i+2, ψ_i+2)] of the Phe‐azaLeu fragment in the model peptide, Boc‐Phe‐azaLeu‐Ala‐OMe, as [(?59^°, 127^°) (107^°, ?4^°)]. This solution conformation supports a βII‐turn structural preference in azaLeu‐containing peptides as predicted by the quantum chemical calculation. Therefore, intercalation of the azaamino acid residue into the i + 2 position in synthetic peptides is expected to provide a stable β‐turn formation, and this could be utilized in the design of new peptidomimetics adopting a β‐turn scaffold.     

Reengineering a type II β-turn as a potential helix nucleator. Part I. Crystal structure of Boc-Val-Pro-(D)Asp-Asp-Val-OMe monohydrate

The crystal structure of Boc-Val₁-Pro₂-(D)Asp₃-Asp₄-Val₅-OMe is described as a type II β-turn reengineered into a potential helix nucleator. (o)Asp₃ in the peptide is responsible for the configurationally guided LD chiral type n β-turn centered at Pro₂-(D)Asp₃, as well as the partially developed LL chiral type I β-turn centered at Asp₄-Val₅ by acceptance of a conformation nucleating H-bond from Val₅NH to its carboxylic oxygen.     

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.     

Conformationally restrained peptides: crystal structure of tert-butyloxycarbonyl-L-alanyl-D-alanyl-D-aminosuccinyl-glycyl-L-alanine methyl ester

The solid-state structure of a heterochiral peptide embodying a D-aminosuccinyl peptide (D-ASU) and a D-Ala was studied in order to analyse the effects of Asu and amino acids with inverse chirality on peptide conformation. The crystal structure has been determined by X-ray diffraction techniques and refined to a final R factor of 0.043. The molecule adopts an unusual overall 'S-shape’ conformation due to two consecutive type II β-turns. In this molecule it is possible to compare a type II β-bend conformation (L-Ala¹-D-Ala²) favoured by the presence of a D-residue at second corner to a type II β-turn (D-Asu³-Gly⁴) favoured by the presence of a D-ASU residue at first corner. In agreement with previous studies, this structure confirms that the Asu has a high propensity to adopt a type II or II′β-bend conformation and that it may be used as a strong determinant of these structural motifs. © Munksgaard 1996.     

Peptide mimics for structural features in proteins

The crystal structure determination of three heptapeptides containing α-aminoisobutyryl (Aib) residues as a means of helix stabilization provides a high-resolution characterization of 6→1 hydrogen-bonded conformations, reminiscent of helix-terminating structural features in proteins. The crystal parameters for the three peptides, Boc-Val-Aib-X-Aib-Ala-Aib-Y-OMe, where X and Y are Phe, Leu (I), Leu, Phe (II) and Leu, Leu (III) are: (I) space group P1, Z= 1, a= 9.903 Å, b 10.709 Å, c= 11.969 Å, α= 102.94°, β=103.41°, γ= 92.72°, R= 4.55%; (II) space group P2₁, Z= 2, a=10.052 Å, b=17.653 Å, c= 13.510 Å, β=108.45°. R= 4.49%; (III) space group P1, Z= 2 (two independent molecules IIIa and IIIb in the asymmetric unit). a=10.833 Å, b=13.850 Å, c=16.928 Å, α=99.77°, β=105.90°, γ= 90.64°, R= 8.54%. In all cases the helices form 3_10/α-helical (or 3l₁₀-helical) structures, with helical columns formed by head-to-tail hydrogen bonding. The helices assemble in an all-parallel motif in crystals I and III and in an antiparallel motif in II. In the four crystallographically characterized molecules, I, II, IIIa and IIIb, Aib(6) adopts a left-handed helical (ht) conformation with positive φψ values, resulting in 6→1 hydrogen-bond formation between Aib(2) CO and Leu(7)/Phe(7) NH groups. In addition a 4→1 hydrogen bond is seen between Aib(3) CO and Aib(6) NH groups. This pattern of hydrogen bonding is often observed at the C-terminus of helices in proteins, with the terminal π-type turn being formed by four residues adopting the h_Rh_Rh_Rh_L conformation.     

Synthesis and solution conformation of c(D-Trp-D-Cys(SO3-Na+)-Pro-D-Val-Leu), a potent endothelin-A receptor antagonist

The endothelin family of polypeptides are known to exert potent physiological effects which include cardiovascular regulation. The solution conformation and dynamics of c(D-Trp-D-Cys(SO₃-Na⁺)-Pro-D-Val-Leu), a potent endothelin-A receptor-selective antagonist, were characterized in aqueous solution by NMR spectroscopy and molecular modeling. NMR-derived conformational constraints were combined with computer-assisted molecular modeling using distance geometry calculations and energy minimization. The pentapeptide backbone is shown to adopt a single conformation in solution comprising a type II β-turn and an inverse γ-turn, with each residue in the trans conformation. Molecular dynamics were explored using relaxation measurements and low-temperature studies, and indicate that the peptide backbone is highly constrained with little conformational mobility present.     

Synthesis and solution structure of an S-glycosylated cyclic hexapeptide

Synthesis and conformational analysis of the S-glycosylated cyclic hexapeptide cyclo(-d -Pro¹-Phe²-Cys³(tetra-O-acetyl-β-d -galactopyranosyl)-Trp⁴-Lys(Z)⁵-Phe⁶-) I was carried out to examine the influence of a saccharide residue in position i of a standard β-turn on the formation of reverse turns and on the biological activity. Synthesis was carried out in the liquid phase employing a galactosylated cysteine building block. The cyclization reagents DPPA/NaHCO₃ avoided high dilution conditions. Spectroscopic data were extracted from homo- and heteronuclear 2D-NMR techniques (TOCSY, NOESY, HMQC, HMQC-TOCSY, HMBCS-270). For structural refinement restrained molecular dynamics (MD) simulations in vacuo and with explicit DMSO as solvent were performed. Finally, simulations in DMSO without experimental restraints provided insight in stability and dynamics of the structural model. A comparison of the S-glycosylated Cys³ peptide with the analogous Thr³ peptide exhibits a similar overall conformation of the hexapeptide [βII’d -Pro-Phe and another β-turn about Trp⁴-Lys⁵(Z)]. However, the latter shows a distinct dynamic flip βI, βII in the glycopeptide, whereas the Thr-analogue only populates βI. This influence is attributed to a βI stabilizing effect of a hydrogen bridge of Thr-O, in position i to the NH of the amino acid in position i+ 2, which is lacking in the glycosylated compound.     

Proline-containing β-turns

The conformations of the dipeptide t-Boc-Pro-d Ala-OH and the tripeptide tBoc-Pro-d Ala-Ala-OH have been determined in the crystalline state by X-ray diffraction and in solution by CD, n.m.r. and i.r. techniques. The unit cell of the dipeptide crystal contains two independent molecules connected by intermolecular hydrogen bonds. The urethane-proline peptide bond is in the cis orientation in both the molecular forms while the peptide bond between Pro and d Ala is in the trans orientation. The single dipeptide molecule exhibits a “bent” structure which approximates to a partial β-turn. The tripeptide adopts the 4 → 1 hydrogen-bonded type II β-turn with all trans peptide bonds. In solution, the CD and i.r. data on the dipeptide indicate an ordered conformation with an intramolecular hydrogen bond. N.m.r. data indicate a significant proportion of the conformer with a trans orientation at the urethane-proline peptide bond. The temperature coefficient of the amide proton of this conformer in DMSO-d₆ points to a 3 → 1 intramolecular hydrogen bond. Taken together, the data on the dipeptide in solution indicate the presence (in addition to the cis conformer) of a C₇ conformation which is absent in the crystalline state. The spectral data on the tripeptide indicate the presence of the type II β-turn in solution in addition to the nonhydrogen-bonded conformer with the cis peptide bond between the urethane and proline residues. The relevance of these data to studies on the substrate specificity of collagen prolylhydroxylase is pointed out.