Conformational effects of C(alpha,alpha)-dipropargylglycine as a constrained residue

A useful synthon to approach artificial phenylalanyl peptides in a [2 + 2 + 2] cycloaddition reaction, C(alpha,alpha)-dipropargylglycine (Dprg) is examined for its conformational preferences as a constrained residue. Crystal structure analysis and preliminary NMR results establish possible preference of the residue for folded (alpha) rather than extended (beta) region of the straight phi,psi conformational space. Boc-Dprg-L-Leu-OMe (1) displays two molecular conformations within the same crystallographic asymmetric unit, with Dprg in the alpha(R) or alpha(L) conformation, participating in a type I beta-turn or an alpha(L)-alpha(R)-type fold, in which Leu(2) assumes the alpha(R) conformation stereochemically favored for an L-chiral residue. Boc-Dprg-D-Val-L-Leu-OMe (2) displays a type I' beta-turn conformation in crystal, with both Dprg(1) and D-Val(2) assuming the alpha(L) conformation stereochemically favored for a D-chiral residue, with 4 --> 1 type hydrogen bond linking L-Leu(3) NH with Boc CO. NMR analysis using temperature variation, solvent titration, and a spin probe study suggests a fully solvent-exposed nature of Dprg NH, ruling out a fully extended C(5)-type conformation for this residue, and solvent sequestered nature of L-Leu(3) NH, suggesting possibility of a beta-turn due to Dprg assuming a folded conformation.     

Structural recognition of an ICAM-1 peptide by its receptor on the surface of T cells: conformational studies of cyclo (1, 12)-Pen-Pro-Arg-Gly-Gly-Ser-Val-Leu-Val-Thr-Gly-Cys-OH.

The purpose of this study is to elucidate the solution conformation of cyclic peptide 1 (cIBR), cyclo (1, 12)-Pen1-Pro2-Arg3-Gly4-Gly5-Ser6-Val7-Leu8-V al9-Thr10-Gly11-Cys12-OH, using NMR, circular dichroism (CD) and molecular dynamics (MD) simulation experiments. cIBR peptide (1), which is derived from the sequence of intercellular adhesion molecule-1 (ICAM-1, CD54), inhibits homotypic T-cell adhesion in vitro. The peptide hinders T-cell adhesion by inhibiting the leukocyte function-associated antigen-1 (LFA-1, CD11a/CD18) interaction with ICAM-1. Furthermore, Molt-3 T cells bind and internalize this peptide via cell surface receptors such as LFA-1. Peptide internalization by the LFA-1 receptor is one possible mechanism of inhibition of T-cell adhesion. The recognition of the peptide by LFA-1 is due to its sequence and conformation; therefore, this study can provide a better understanding for the conformational requirement of peptide-receptor interactions. The solution structure of 1 was determined using NMR, CD and MD simulation in aqueous solution. NMR showed a major and a minor conformer due to the presence of cis/trans isomerization at the X-Pro peptide bond. Because the contribution of the minor conformer is very small, this work is focused only on the major conformer. In solution, the major conformer shows a trans-configuration at the Pen1-Pro2 peptide bond as determined by HMQC NMR. The major conformer shows possible beta-turns at Pro2-Arg3-Gly4-Gly5, Gly5-Ser6-Val7-Leu8, and Val9-Thr10-Gly11-Cys12. The first beta-turn is supported by the ROE connectivities between the NH of Gly4 and the NH of Gly5. The connectivities between the NH of Ser6 and the NH of Val7, followed by the interaction between the amide protons of Val7 and Leu8, support the presence of the second beta-turn. Furthermore, the presence of a beta-turn at Val9-Thr10-Gly11-Cys12 is supported by the NH-NH connectivities between Thr10 and Gly11 and between Gly11 and Cys12. The propensity to form a type I beta-turn structure is also supported by CD spectral analysis. The cIBR peptide (1) shows structural similarity at residues Pro2 to Val7 with the same sequence in the X-ray structure of D1-domain of ICAM-1. The conformation of Pro2 to Val7 in this peptide may be important for its binding selectivity to the LFA-1 receptor.     

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

The crystal structures of four peptides incorporating 1-aminocycloheptane-1-carboxylic acid (Ac7c) are described. Boc-Aib-Ac7c-NHMe and Boc-Pro-Ac7c-Ala-OMe adopt beta-turn conformations stabilized by an intramolecular 4----1 hydrogen bond, the former folding into a type-I/III beta-turn and the latter into a type-II beta-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 beta-turn conformations in solution.     

Unique side chain conformation of a Leu residue in a triple-helical structure

Single crystal structures of host-guest peptides, (Pro-Hyp-Gly)(4)-Leu-Hyp-Gly-(Pro-Hyp-Gly)(5) (LOG1) and (Pro-Hyp-Gly)(4)- (Leu-Hyp-Gly)(2)-(Pro-Hyp-Gly)(4) (LOG2), have been determined at 1.6 A and 1.4 A resolution, respectively. In these crystals, the side chain conformations of the Leu residues were (+)gauche-trans. This conformational preference for the Leu side chain in the Leu-Hyp-Gly sequence was explained by stereochemical considerations together with statistical analysis of Protein Data Bank data. In the (+)gauche-trans conformation, the Leu side chain can protrude along the radial direction of the rod-like triple-helical molecule. One strong hydrophobic interaction of the Leu residue was observed between adjacent molecules in the LOG2 crystal. Because the Leu-Hyp-Gly sequence is one of the most frequently occurring triplets in Type I collagen, this strong hydrophobic interaction can be expected in a fibrillar structure of native collagen. All the Leu residues in the asymmetric unit of the LOG1 and LOG2 crystals had water molecules hydrogen bonded to their NH. These water molecules made three additional hydrogen bonds with the Hyp OH, the Gly O[double bond]C, and a water molecule in the second hydration shell, forming a tetrahedral coordination of hydrogen bonds, which allows a smaller mean-square displacement factor of this water oxygen atom than those of other water molecules. These hydrogen bonds stabilize the molecular and packing structures by forming one O[double bond]C(Gly)---W---OH(Hyp) intra-molecular linkage and two NH(Leu)---W---O[double bond]C(Gly) and NH(Leu)---W---OH(Hyp) inter-molecular linkages.     

Aromatic interactions in tryptophan-containing peptides: crystal structures of model tryptophan peptides and phenylalanine analogs.

The crystal structures of the peptides, Boc-Leu-Trp-Val-OMe (1), Ac-Leu-Trp-Val-OMe (2a and 2b), Boc-Leu-Phe-Val-OMe (3), Ac-Leu-Phe-Val-OMe (4), and Boc-Ala-Aib-Leu-Trp-Val-OMe (5) have been determined by X-ray diffraction in order to explore the nature of interactions between aromatic rings, specifically the indole side chain of Trp residues. Peptide 1 adopts a type I beta-turn conformation stabilized by an intramolecular 4-->1 hydrogen bond. Molecules of 1 pack into helical columns stabilized by two intermolecular hydrogen bonds, Leu(1)NH...O(2)Trp(2) and IndoleNH...O(1)Leu(1). The superhelical columns further pack into the tetragonal space group P4(3) by means of a continuous network of indole-indole interactions. Peptide 2 crystallizes in two polymorphic forms, P2(1) (2a) and P2(1)2(1)2(1) (2b). In both forms, the peptide backbone is extended, with antiparallel beta-sheet association being observed in crystals. Extended strand conformations and antiparallel beta-sheet formation are also observed in the Phe-containing analogs, Boc-Leu-Phe-Val-OMe (3) and Ac-Leu-Phe-Val-OMe (4). Peptide 5 forms a short stretch of 3(10)-helix. Analysis of aromatic-aromatic and aromatic-amide interactions in the structures of peptides, 1, 2a, 2b are reported along with the examples of 14 Trp-containing peptides from the Cambridge Crystallographic Database. The results suggest that there is no dramatic preference for a preferred orientation of two proximal indole rings. In Trp-containing peptides specific orientations of the indole ring, with respect to the preceding and succeeding peptide units, appear to be preferred in beta-turns and extended structures.     

Conformation analysis of the N-glycosylation site Asn-X-Thr/Ser in glycoproteins

Theoretical conformational analysis of oligopeptides CH3CO-Asn-X-Thr-NHCH3 (X = Gly, Ala, Pro), modelling N-glycosylation site, and their glycosylated derivatives CH3CO-(GlcNAc beta 1-4GlcNAc beta 1) Asn-X-Thr-NHCH3 has been carried out. Active conformations of the site are found, corresponding to structural prerequisities of N-glycosylation: Asn residue's position in beta-turn and hydrogen bond formation between side chains of Asn and Thr/Ser residues. In this case the L conformation of the central residue X is most probable. Since Pro residue does not possess this conformation, sequences with X = Pro are not glycosylated. It is shown that glycosylation of the above-mentioned sites is accompanied by reorientation of the Asn residue's side chains.     

Conformational studies of two bradykinin antagonists by using two-dimensional NMR techniques and molecular dynamics simulations

The solution conformations of two potent antagonists of bradykinin (Arg1-Pro2-Pro3-Gly4-Phe5-Ser6-Pro7-Phe8-Arg9), [Aca(-1),DArg0,Hyp3,Thi5,DPhe7,(N-Bzl)Gly8]BK (1) and [Aaa(-1),DArg0,Hyp3,Thi5,(2-DNal)7,Thi8]BK (2), were studied by using 2D NMR spectroscopy in DMSO-d6 and molecular dynamics simulations. The NMR spectra of peptide 1 reveals the existence of at least two isomers arising from isomerization across the DPhe7-(N-Bzl)Gly8 peptide bond. The more populated isomer possesses the cis peptide bond at this position. The ratio of cis/trans isomers amounted to 7:3. With both antagonists, the NMR data indicate a beta-turn structure for the Hyp3-Gly4 residues. In addition, for peptide 2, position 2,3 is likely to be occupied by turn-like structures. The cis peptide bond between DPhe7 and (N-Bzl)Gly8 in analogue 1 suggests type VI beta-turn at position 7,8. The molecular dynamics runs were performed on both peptides in DMSO solution. The results indicate that the structure of peptide 1 is characterized by type VIb beta-turn comprising residues Ser6-Arg9 and the betaI or betaII-turn involving the Pro2-Thi5 fragment, whereas peptide 2 shows the tendency towards the formation of type I beta-turn at position 2,3. The structures of both antagonists are stabilized by a salt bridge between the guanidine moiety of Arg1 and the carboxyl group of Arg9. Moreover, the side chain of DArg0 is apart of the rest of molecule and is not involved in structural elements except for a few calculated structures.     

Conformational choice at alpha,alpha-di-n-propylglycine residues: helical or fully extended structures?

The conformational analysis of peptides containing a single alpha, alpha-di-n-propylglycine (Dpg) residue incorporated into valine-rich sequences has been undertaken in order to delineate the possible role of sequence effects in stabilizing fully extended (C(5)) or local helical conformations at this residue. The three peptides Boc-Val-Dpg-Val-OMe (3), Boc-Val-Val-Dpg-Val-OMe (4), Boc-Val-Val-Dpg-Val-Val-OMe (5), have been studied by (1)H-nmr methods in chloroform (CDCl(3)) and dimethylsulfoxide (DMSO) solutions. Even in a relatively poorly solvating medium like CDCl(3), all the valine NH groups appear to be solvent-exposed, suggesting an absence of folded beta-turn conformations. However, in both CDCl(3) and DMSO the Dpg NH groups in all the three peptides appear to behave like apparently solvent-inaccessible groups. In fully extended C(5) conformations, the proximity of the NH and CO groups of Dpg may preclude effective solvation due to a combination of stereoelectronic factors. Nuclear Overhauser effects provide support for the largely extended backbones. The crystal structure of peptide 3 reveals an extended conformation at Dpg (2) with straight phi = -176 degrees, psi = 180 degrees. A correlation between the crystallographically observed backbone conformation and solution nmr parameters in DMSO has been attempted using available data. Dpg residues placed in poor helix stabilizing environments may be expected to favor a local C(5) conformation.     

Depsipeptide analogues of elastin repeating sequences: conformational analysis

In this work the effect of elimination of a specific hydrogen bond on the conformation of the repeating peptides of elastin was studied. These repeating sequences are the pentapeptide Val-Pro-Gly-Val-Gly and the hexapeptide Val-Ala-Pro-Gly-Val-Gly. These sequences have been proposed to occur in a beta-turn conformation with a hydrogen bond involving the amide NH of the internal valine residue and the carbonyl oxygen of the residue preceding proline. In the depsipeptide analogues studied in this work, this 4-1 beta-turn hydrogen bond cannot occur. We studied the depsipeptide sequences Val-Pro-Gly-Hiv-Gly and Val-Ala-Pro-Gly-Hiv-Gly (Hiv denotes S-alpha-hydroxyisovaleric acid, the hydroxy acid analogue of valine), as well as the peptide sequences Val-Pro-Gly-Val-Gly and Val-Ala-Pro-Gly-Val-Gly. Compounds studied included sequences with the Boc and benzyl ester protecting groups, derivatives with the acetyl and N-methylamide end groups and polymers of the above sequences. Our conclusions are based on a comparison of depsipeptides with analogous peptides. Conformational analysis was carried out by nmr, CD, and ir spectroscopy. We propose that in the repeating sequences of elastin an equilibrium exists between a gamma-turn structure and a beta-turn structure in the Pro-Gly segment resulting in a structure that combines flexibility with strong conformational preferences. The C7 involves the amide NH of the internal glycine and the carbonyl oxygen of the residue preceding proline. In the N-methylamide derivatives a similar equilibrium exists in the Gly-Val-Gly segment. In the depsipeptides the beta-turn cannot occur and only the gamma-turn is seen. In the polydepsipeptides the major conformational feature is a type I beta-turn involving Gly5 NH and Pro CO.     

Role of peptide backbone conformation on biological activity of chemotactic peptides

To investigate the role of peptide backbone conformation on the biological activity of chemotactic peptides, we synthesized a unique analog of N-formyl-Met-Leu-Phe-OH incorporating the C alpha,alpha disubstituted residue, dipropylglycine (Dpg) in place of Leu. The conformation of the stereochemically constrained Dpg analog was examined in the crystalline state by x-ray diffraction and in solution using NMR, IR, and CD methods. The secretagogue activity of the peptide on human neutrophils was determined and compared with that of a stereochemically constrained, folded type II beta-turn analog incorporating 1-aminocyclohexanecarboxylic acid (Ac6c) at position 2 (f-Met-Ac6c-Phe-OMe), the parent peptide (f-Met-Leu-Phe-OH) and its methyl ester derivative (f-Met-Leu-Phe-OMe). In the solid state, the Dpg analog adopts an extended beta-sheet-like structure with an intramolecular hydrogen bond between the NH and CO groups of the Dpg residue, thereby forming a fully extended (C5) conformation at position 2. The phi and psi values for Met and Phe residues are significantly lower than the values expected for an ideal antiparallel beta conformation causing a twist in the extended backbone both at the N and C termini. Nuclear magnetic resonance studies suggest the presence of a significant population of the peptide molecules in an extended antiparallel beta conformation and the involvement of Dpg NH in a C5 intramolecular hydrogen bond in solutions of deuterated chloroform and deuterated dimethyl sulfoxide. IR studies provide evidence for the presence of an intramolecular hydrogen bond in the molecule and the antiparallel extended conformation in chloroform solution. CD spectra in methanol, trifluoroethanol, and trimethyl phosphate indicate that the Dpg peptide shows slight conformational flexibility, whereas the folded Ac6c analog is quite rigid. The extended Dpg peptide consistently shows the highest activity in human peripheral blood neutrophils, being approximately 8 and 16 times more active than the parent peptide and the folded Ac6c analog, respectively. However, the finding that all four peptides have ED50 (the molar concentration of peptide to induce half-maximal enzyme release) values in the 10(-8)-10(-9) M range suggests that an induced fit mechanism may indeed be important in this ligand-receptor interaction. Moreover, it is also possible that alterations in the backbone conformation at the tripeptide level may not significantly alter the side chain topography and/or the accessibility of key functional groups important for interaction with the receptor.     

Conformational analysis of cyclolinopeptide A, a cyclic nonapeptide: nuclear Overhauser effect and energy minimization studies

The conformation of cyclolinopeptide A [cyclo(Pro-Pro-Phe-Phe-Leu-Ile-Ile-Leu-Val)], a naturally occurring cyclic nonapeptide has been investigated in dimethylsulfoxide solution by 270 MHz 1H-nmr. A complete assignment of all C alpha H and NH resonances has been accomplished using two-dimensional correlated spectroscopy and nuclear Overhauser effects (NOEs). Analysis of interresidue NOEs and JHNC alpha H values permit construction of a molecular model for the cyclic peptide backbone. The crude model derived from nmr has been used as a starting point for energy minimization, which yields a refined structure largely compatible with nmr observations. The major features of the conformation of cyclolinopeptide A are a Type VI beta-turn centered at Pro(1)-Pro(2), with a cis peptide bond between these residues and a gamma-turn (C7 structure) centered at Ile(6). Two intramolecular hydrogen bonds Val(9) CO--Phe(3)NH (4----1) and Leu(5) CO--Ile(7)NH (3----1) are observed in the low-energy conformation. The limited solvent accessibility observed for the Val(9) and Leu(5) NH groups in the nmr studies are rationalized in terms of steric shielding.     

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

Two isomeric, acyclic tetrapeptides containing a Z-dehydrophenylalanine residue (delta Z-Phe) at position 2 or 3, Boc-Leu-Ala-delta Z-Phe-Leu-OMe (1) and Boc-Leu-delta Z-Phe-Ala-Leu-OMe (2), have been synthesized and their solution conformations investigated by 270 MHz 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 (Ci alpha H----Ni+1H and NiH----Ni+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-delta Z-Phe- Type II beta-turn structure and a second species with delta Z-Phe adopting a partially extended conformation with psi values of +/- 100 degrees to +/- 150 degrees. In peptide 2 the results are compatible with an equilibrium between a highly folded consecutive beta-turn structure for the -Leu-delta Z-Phe-Ala- segment and an almost completely extended conformation.     

Role of azaamino acid residue in beta-turn formation and stability in designed peptide.

The structural perturbation induced by C(alpha)-->N(alpha) 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 beta-turn motif with a dihedral angle of phi= +/- 90 degrees, psi=0 degrees. This suggests that incorporation of the azaXaa residue into the i+2 position of designed peptides could stabilize the beta-turn structure. The model azaLeu-containing peptide, Boc-Phe-azaLeu-Ala-OMe, which is predicted to adopt a beta-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 [ (phi i+1, psi i+1) (phi i+2, psi i+2)] of the Phe-azaLeu fragment in the model peptide, Boc-Phe-azaLeu-Ala-OMe, as [(-59 degrees, 127 degrees) (107 degrees, -4 degrees)]. This solution conformation supports a betaII-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 beta-turn formation, and this could be utilized in the design of new peptidomimetics adopting a beta-turn scaffold.     

Solid state and solution conformations of a hybrid alphagammaalphaalphagammaalpha hexapeptide. Characterization of a backbone expanded analog of the alpha-polypeptide 3(10)-helix

The stereochemically constrained gamma amino acid residue gabapentin (1-(aminomethyl)cyclohexaneacetic acid, Gpn) has been incorporated into a host alpha-peptide sequence. The structure of a hybrid alphagammaalphaalphagammaalpha peptide, Boc-Leu-Gpn-Aib-Leu-Gpn-Aib-OMe in crystals reveals a continuous helical conformation stabilized by three intramolecular 4 --> 1 C(12) hydrogen bonds across the alphagamma/alphagamma segments and one C(10) hydrogen bond across the central alphaalpha segment. This conformation corresponds to an expanded analog of the canonical all-alpha polypeptide 3(10)-helix, with insertion of two additional backbone atoms at each gamma residue. Solvent dependence of NH chemical shifts in CDCl(3) solution are consistent with conformation in which the NH groups of Aib (3), Leu (4), Gpn (5), and Aib (6) are hydrogen bonded, a feature observed in the solid state. The nonsequential NOEs between Gpn (2) NH <--> Leu (4) NH and Gpn (2) NH <--> Gpn (5) NH support the presence of additional conformations in solution. Temperature-dependent line broadening of NH resonances confirms the occurrence of rapid exchange between multiple conformations at room temperature. Two conformational models which rationalize the observed nonsequential NOEs are presented, both of which contain three hydrogen bonds and are consistent with the known stereochemical preferences of the Gpn residue.     

Conformation of di-n-propylglycine residues (Dpg) in peptides: crystal structures of a type I' beta-turn forming tetrapeptide and an alpha-helical tetradecapeptide.

The crystal structures of two oligopeptides containing di-n-propylglycine (Dpg) residues, Boc-Gly-Dpg-Gly-Leu-OMe (1) and Boc-Val-Ala-Leu-Dpg-Val-Ala-Leu-Val-Ala-Leu-Dpg-Val-Ala-Leu-OMe (2) are presented. Peptide 1 adopts a type I'beta-turn conformation with Dpg(2)-Gly(3) at the corner positions. The 14-residue peptide 2 crystallizes with two molecules in the asymmetric unit, both of which adopt alpha-helical conformations stabilized by 11 successive 5 --> 1 hydrogen bonds. In addition, a single 4 --> 1 hydrogen bond is also observed at the N-terminus. All five Dpg residues adopt backbone torsion angles (phi, psi) in the helical region of conformational space. Evaluation of the available structural data on Dpg peptides confirm the correlation between backbone bond angle N-C(alpha)-C' (tau) and the observed backbone phi,psi values. For tau > 106 degrees, helices are observed, while fully extended structures are characterized by tau < 106 degrees. The mean tau values for extended and folded conformations for the Dpg residue are 103.6 degrees +/- 1.7 degrees and 109.9 degrees +/- 2.6 degrees, respectively.     

Influence of N-terminal residue stereochemistry on the prolyl amide geometry and the conformation of 5-tert-butylproline type VI beta-turn mimics.

The effects of N-terminal amino acid stereochemistry on prolyl amide geometry and peptide turn conformation were investigated by coupling both L- and D-amino acids to (2S, 5R)-5-tert-butylproline and L-proline to generate, respectively, N-(acetyl)dipeptide N'-methylamides 1 and 2. Prolyl amide cis- and trans-isomers were, respectively, favored for peptides 1 and 2 as observed by proton NMR spectroscopy in water, DMSO and chloroform. The influence of solvent composition on amide proton chemical shift indicated an intramolecular hydrogen bond between the N'-methylamide proton and the acetamide carbonyl for the major conformer of dipeptides (S)-1, that became less favorable in (R)-1 and 2. The coupling constant (3J(NH,alpha)) values for the cis-isomer of (R)-1 indicated a phi2 dihedral angle value characteristic of a type VIb beta-turn conformation in solution. X-ray crystallographic analysis of N-acetyl-D-leucyl-5-tert-butylproline N'-methylamide (R)-lb showed the prolyl residue in a type VIb beta-turn geometry possessing an amide cis-isomer and psi3-dihedral angle having a value of 157 degrees, which precluded an intramolecular hydrogen bond. Intermolecular hydrogen bonding between the leucyl residues of two turn structures within the unit cell positioned the N-terminal residue in a geometry where their phi2 and psi2 dihedral angle values were not characteristic of an ideal type VIb turn. The circular dichroism spectra of tert-butylprolyl peptides (S)- and (R)-1b were found not to be influenced by changes in solvent composition from water to acetonitrile. The type B spectrum exhibited by (S)-1b has been previously assigned to a type VIa beta-turn conformation [Halab L, Lubell WD. J. Org. Chem. 1999; 64: 3312-3321]. The type C spectrum exhibited by the (R)-lb has previously been associated with type II' beta-turn and alpha-helical conformations in solution and appears now to be also characteristic for a type VIb geometry.     

Stability of protein-bound conformations of bioactive peptides: the folded conformation of an epidermal growth factor-like thrombomodulin fragment is similar to that recognized by thrombin

The relationship between the free and bound conformations of bioactive peptides is explored using the epidermal growth factor (EGF)-like thrombomodulin fragment hTM409-426 as a model system. The hTM409-426 peptide has a sequence of C(409)PEGYILDDGFIC(421)TDIDE (with a disulfide bond between Cys409 and Cys421) and is a selective inhibitor of thrombin. Upon binding to thrombin, hTM409-426 adopts a well-defined conformation-namely, a beta-turn followed by an antiparallel beta-sheet, similar to those found in all other EGF-like protein repeats (Hrabal et al., Protein Science, 1996, Vol. 5, 195-203). Here we demonstrate that, at pH 6.8 and at 25 degrees C, the hTM409-426 peptide in the free state is very flexible, but still populates a type II beta-turn over residues Pro410-Glu411-Gly412-Tyr413 and the clustering of some hydrophobic side chains, both of which are present in the thrombin-bound conformation. At a lower temperature of 5 degrees C, significant conformational shifts of the C alpha H proton resonances and extensive medium- and long-range NOEs are observed, indicating the presence of folded conformations with unique backbone-backbone and side-chain interactions. A comparison of the NOE patterns in the free state with transferred NOEs shows that the free-state folded and the thrombin-bound conformations of the hTM409-426 peptide are very similar, particularly over residues Pro410-Ile424. The folded conformation of hTM409-426 appears to be stabilized by two hydrophobic clusters, one formed by the side chains of residues Pro410, Tyr413, Leu415, and Phe419 and the Cys409-Cys421 disulfide bond, the second involving residues Ile414 and Ile424. These results indicate that the overall topology of the thrombin-bound conformation of the hTM409-426 peptide is prefolded in the free state and the primary sequence (including the disulfide bond) may be selective for an ensemble of conformations similar to that recognized by thrombin.     

The role of hydroxyproline in collagen folding: conformational energy calculations on oligopeptides containing proline and hydroxyproline

We have observed that the rate of folding of the enzymatically hydroxylated form of poly(Gly-Pro-Pro) into the triple-helical conformation is considerably higher than that of the unhydroxylated polypeptide [R. K. Chopra and V. S. Ananthanarayanan (1982) Proc. Natl. Acad. Sci. USA 79 , 7180–7184]. In this study, we examine a plausible kinetic pathway for triple-helix formation by selecting peptide models for the unhydroxylated collagen molecule, and computing their conformational energies before and after proline hydroxylation. Starting with the available data on the preferred conformations of proline- and hydroxyproline-containing peptide sequences, energy minimization was carried out on the following pairs of peptides: Gly-Ala-Pro-Gly-Ala and Gly-Ala-Hyp-Gly-Ala; Gly-Pro-Pro-Gly-Ala and Gly-Pro-Hyp-Gly-Ala; Gly-Ala-Pro-Gly-Ala-Pro and Gly-Ala-Hyp-Gly-Ala-Hyp. It was found that, with each pair of peptides, the energetically most favorable conformation (I) has an extended structure at the Gly-Ala or Gly-Pro segment and a β-bend at the Pro-Gly or Hyp-Gly segment. In the Hyp-containing peptides, this conformation is further stabilized by a (Hyp_{i + 2})OH…OC(Gly_i) hydrogen bond. Conformation I is lower in energy by about 6–13 kcal/mol of the peptide than the fully extended conformations that resemble the single collagen polypeptide chain and contain no intramolecular hydrogen bond. In contrast to the proline counterpart, the hydroxyproline-containing peptides are found capable of adopting a partially extended conformation that does not contain the β-bend but retains the (Hyp)OH…OC(Gly) hydrogen bond. The energy of this conformation is intermediate between conformation I and the fully extended conformation. The continuation of the β-bend along the chain is restricted by stereochemical constraints that are more severe in the latter two pairs of peptides than in the first pair. Such a restriction may be considered to trigger the “unbending” of the minimum energy conformation leading to its straightening into the fully extended conformation; the latter, in turn, would lead to triple-helix formation through favorable interchain interactions. We propose that the partially extended conformation in the Hyp-containing peptides could serve as a kinetic intermediate on the way to forming the fully extended conformation. Because of the (Hyp_{i + 2})OH…OC(Gly_i) hydrogen bond, this conformation would also serve to lock the trans geometry at the Gly-Ala(Pro) and Ala(Pro)-Hyp peptide bonds, thereby enhancing the rate of their helix formation. A scheme for collagen folding in proposed on the basis of these results.     

Design of peptides with branched beta-carbon dehydro-residues: syntheses, crystal structures and molecular conformations of two peptides, (I) N-Carbobenzoxy-DeltaVal-Ala-Leu-OCH3 and (II) N-Carbobenzoxy-DeltaIle-Ala-Leu-OCH3.

Highly specific structures can be designed by inserting dehydro-residues into peptide sequences. The conformational preferences of branched beta-carbon residues are known to be different from other residues. As an implication it was expected that the branched beta-carbon dehydro-residues would also induce different conformations when substituted in peptides. So far, the design of peptides with branched beta-carbon dehydro-residues at (i + 1) position has not been reported. It may be recalled that the nonbranched beta-carbon residues induced beta-turn II conformation when placed at (i + 2) position while branched beta-carbon residues induced beta-turn III conformation. However, the conformation of a peptide with a nonbranched beta-carbon residue when placed at (i + 1) position was not found to be unique as it depended on the stereochemical nature of its neighbouring residues. Therefore, in order to induce predictably unique structures with dehydro-residues at (i + 1) position, we have introduced branched beta-carbon dehydro-residues instead of nonbranched beta-carbon residues and synthesized two peptides: (I) N-Carbobenzoxy-DeltaVal-Ala-Leu-OCH3 and (II) N-Carbobenzoxy-DeltaIle-Ala-Leu-OCH3 with DeltaVal and DeltaIle, respectively. The crystal structures of peptides (I) and (II) have been determined and refined to R-factors of 0.065 and 0.063, respectively. The structures of both peptides were essentially similar. Both peptides adopted type II beta-turn conformations with torsion angles; (I): phi1 = -38.7 (4) degrees, psi1 = 126.0 (3) degrees; phi2 = 91.6 (3) degrees, psi2 = -9.5 (4) degrees and (II): phi1 = -37.0 (6) degrees, psi1 = 123.6 (4) degrees, phi2 = 93.4 (4), psi2 = -11.0(4) degrees respectively. Both peptide structures were stabilized by intramolecular 4-->1 hydrogen bonds. The molecular packing in both crystal structures were stabilized in each by two identical hydrogen bonds N1...O1' (-x, y + 1/2, -z) and N2...O2' (-x + 1, y + 1/2, -z) and van der Waals interactions.     

CD-n.m.r. study of the solution conformation of bradykinin analogs containing alpha-aminoisobutyric acid

The conformation in aqueous solution of several alpha-aminoisobutyric acid (AIB)-containing analogs of bradykinin (BK) has been probed by complementary CD and 1H n.m.r. measurements. The conclusion reached is that substitution of AIB for Pro2 and/or Pro3 in BK stabilizes a degree of beta-turn conformation in the N-terminal tetrapeptide moiety of the resulting analogs. Changing the solvent from water to DMSO or TFE further enhances the contribution of particular hydrogen bonded structures to the time-averaged conformation of these peptides. Bradykinin and [AIB7]-BK adopt similar hydrogen bonded conformations in TFE, apparently with a contribution from a beta-turn involving their common Arg1-Pro2-Pro3-Gly4 moiety. The contrasting biological activities of BK and its AIB-analogs are considered in terms of the conformational analogy between the AIB-residue and cis' Pro and the propensity for a beta-turn at the N-terminus of the peptide.