Molecular conformation and packing of peptide hairpins in the solid state: Structures of two synthetic octapeptides containing 1-aminocycloalkane-1-carboxylic acid residues at the i+2 position of the βturn

Peptide β-hairpin formation is facilitated by centrally positioned D-Pro-Xxx segments. The synthetic peptides Boc-Leu-Phe-Val-D-Pro-Ac6c-Leu-Phe-Val-OMe (1) and Boc-Leu-Phe-Val-D-Pro-Ac8c-Leu-Phe-Val-OMe (2) were synthesized in order to explore the role of bulky 1-aminocycloalkane-1-carboxylic acid residues (Acnc, where n is the number of carbon atoms in the ring), at the i+2 position of the nucleating β turn in peptide β hairpins. Peptides 1 and 2 crystallize in the monoclinic space group P21 with two molecules in the asymmetric unit. The crystal structures of 1 and 2 provide conformational parameters for four peptide hairpin molecules. In all cases, the central segments adopts a type II′ β-turn conformation, and three of the four possible cross-strand hydrogen bonds are observed. Fraying of the hairpins at the termini is accompanied by the observation of NH...∏ interaction between the Leu(1)NH group and Phe(7) aromatic group. Cross strand stabilizing interactions between the facing residues Phe(2) and Phe(7) are suggested by the observed orientation of aromatic rings. Anomalous far-UV CD spectra observed in solution suggest that close proximity of the Phe rings is maintained even in isolated molecules. In both peptides 1 and 2, the asymmetric unit consists of approximately orthogonal hairpins, precluding the formation of a planar β-sheet arrangement in the solid state. Solvent molecules, one dioxane and one water in 1, three water molecules in 2, mediate peptide association. A comparison of molecular conformation and packing motifs in available β-hairpin structures permits delineation of common features. The crystal structures of β-hairpin peptides provide a means of visualizing different modes of β-sheet packing, which may be relevant in developing models for aggregates of polypeptides implicated in disease situations

Designed peptides with homochiral and heterochiral diproline templates as conformational constraints

Diproline segments have been advanced as templates for nucleation of folded structure in designed peptides. The conformational space available to homochiral and heterochiral diproline segments has been probed by crystallographic and NMR studies on model peptides containing L-Pro-L-Pro and D-Pro-L-Pro units. Four distinct classes of model peptides have been investigated: a) isolated D-Pro-L-Pro segments which form type II β-turn; b) D-Pro-L-Pro-L-Xxx sequences which form type II′-I (βII′-I′, consecutive β-turns) turns; c) D-Pro-L-Pro-D-Xxx sequences; d) L-Pro-L-Pro-L-Xxx sequences. A total of 17 peptide crystal structures containing diproline segments are reported. Peptides of the type Piv-D-Pro-L-Pro-L-Xxx-NHMe are conformationally homogeneous, adopting consecutive β-turn conformations. Peptides in the series Piv-D-Pro-L-Pro-D-Xxx-NHMe and Piv-L-Pro-L-Pro-L-Xxx-NHMe, display a heterogeneity of structures in crystals. A type VIa β-turn conformation is characterized in Piv-L-Pro-L-Pro-L-Phe-OMe (18), while an example of a 5→1 hydrogen bonded α-turn is observed in crystals of Piv-D-Pro-L-Pro-D-Ala-NHMe (11). An analysis of pyrrolidine conformations suggests a preferred proline puckering geometry is favored only in the case of heterochiral diproline segments. Solution NMR studies, reveal a strong conformational influence of the C-terminal Xxx residues on the structures of diproline segments. In L-Pro-L-Pro-L-Xxx sequences, the Xxx residues strongly determine the population of Pro-Pro cis conformers, with an overwhelming population of the trans form in L-Xxx = L-Ala (19)

Conformation of di-n-propylglycine residues (Dpg) in peptides: Crystal structures of a type I′β-turn forming tetrapeptide and an α-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-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 α-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 (φ,ψ) in the helical region of conformational space. Evaluation of the available structural data on Dpg peptides confirm the correlation between backbone bond angle N-Cα-C'(ζ) and the observed backbone φ,ψ, values. For ζ > 106°, helices are observed, while fully extended structures are characterized by ζ < 106°. The mean values for extended and folded conformations for the Dpg residue are 103.6° ± 1.7° and 109.9° ± 2.6°, respectively

INDUCTION OF FOLDED STRUCTURES IN DESIGNED PEPTIDES USING CONFORMATIONALLY CONSTRAINED RESIDUES

Folding into compact globular structures, with well-defined modules of secondary structure, appears to be a characteristic of long polypeptide chains, with a specific patterning of coded amino acid residues along the length of sequence. Cooperative hydrogen bond driven secondary structure formation and solvent forces, which contribute favorably to the entropy of folding, by promoting compaction of the polymeric chain, have long been discussed as major determinants of the folding process. First principles design approaches, which use non-coded amino acids, employ an alternative structure directing strategy, by using amino acid residues which exhibit a strong conformational bias for specific regions of the Ramachandran map. This overview of ongoing studies in the authors' laboratory, attempts to explore the use of conformationally restricted amino acid residues in the design of peptides with well-defined secondary structures. Short peptides composed of 20 genetically coded amino acids usually exist in solution as an ensemble of equilibrating conformations. Apolar peptide sequences, which are readily soluble in organic solvents like chloroform and methanol, facilitate formation of structures which are predominately driven by intramolecular hydrogen bond formation. The choice of sequences containing residues with a limited range of conformational choices strongly favors formation of local turn structures, stabilized by short range intramolecular hydrogen bonds. Two residue beta-turns can nucleate either helical or hairpin folding, depending on the precise conformation of the turn segment Restriction of the conformational space available to amino acid residues is easily achieved by introduction of an additional alkyl group at the C alpha carbon atom or by side chain backbone cyclization, as in proline. Studies of synthetic sequences incorporating two prototype residues alpha-aminoisobutyric acid (Aib) and D-proline (DPro) illustrate the utility of the strategy in construction of helices and hairpins. Extensions to the design of conformationally switchable sequences and structurally defined hybrid peptides containing backbone homologated residues are also surveyed

Aib Residues in Peptaibiotics and Synthetic Sequences: Analysis of Nonhelical Conformations

The \alpha-aminoisobutyric (Aib) residue has generally been considered to be a strongly helicogenic residue as evidenced by its ability to promote helical folding in synthetic and natural sequences. Crystal structures of several peptide natural products, peptaibols, have revealed predominantly helical conformations, despite the presence of multiple helix-breaking Pro or Hyp residues. Survey of synthetic Aib-containing peptides shows a preponderance of $3_{10-},\alpha-$ and mixed $3_{10}/\alpha$-helical structures. This review highlights the examples of Aib residues observed in nonhelical conformations, which fall primarily into the polyproline II (PII) and fully extended regions of conformational space. The achiral Aib residue can adopt both left $\alpha_L$ and right $\alpha_R$ handed helical conformations. In sequences containing chiral amino acids, helix termination can occur by means of chiral reversal at an Aib residue, resulting in formation of a Schellman motif. Examples of Aib residues in unusual conformations are illustrated by surveying a database of Aib-containing crystal structures

Balanced Population Stochastic Variational Inference

The 3_1_0 helix and \alpha helix are closely related secondary structures observed in polypeptides. The 3_1_0 helix is characterized by successive $4\rightarrow1$ (C_1_0) hydrogen bonds (C_1_0= 10 atom hydrogen-bonding ring) of the type $C=O_i\cdot\cdot\cdot$ $HN_{i+3},$ while the $\alpha$ helix displays a hydrogen bond of the type $C= O_i\cdot\cdot\cdot$ $HN_{i+4} (C_{13})$ The $\alpha$ helix is widely distributed in proteins, while the occurrence ofsegments of 3_1_0 helix is very much less frequent. In polypeptides containing $C^{\alpha,\alpha}$ dialkylated residues, $\alpha$-aminoisobutyric acid (Aib) being the prototype, both the 3_1_0 and a helical structures are detected. In homooligomers ofAib , 3_1_0 helices are invariably found in crystals,^[^3^] while in heteromeric sequences the precise helical type appears to depend on both Aib content and positioning. While distinctions between 3_1_0 and \alpha helices are possible in the crystalline state, such differentiation becomes difficult in solution. Circular dichroism (CD) has been proposed for distinguishing between 3_1_0 and \alpha helix structures by using the ratio of CD bands at 222 nm and 207/208 nm. However, the use of the $[\theta]_{222}/[\theta]_{208}$ ratio has been questioned, suggesting that the distinction between 3_1_0 and $\alpha$ helix structures by chiroptical methods may not be readily possible. The conventional interpretation ofthe CD spectra of helical polypeptides has been further called into question by the careful work of Kemp and co-workers, who have reported the observation oflarge values of[\theta]_2_2_2, which are inconsistent with those currently accepted for 100% helical structures. Kemp et al. have noted that the 222 nm $n\pi$* band has not been modeled satisfactorily by heory. The widespread use ofthe CD band intensities at 208 and 222 nm, in estimating helicity values quantitatively and in making qualitative distinctions between helix subtypes, underscores the importance importance ofrelating CD spectral intensities to specific peptide structural features. In addressing this issue, Dang and Hirst have used an improved theoretical method to calculate the 220 nm CD band intensities and have suggested that [\theta]_2_2_0 is extremely sensitive to main-chain hydrogen-bond length. They argue that “shortening from a conventional oxygen– nitrogen separation ofabout $3.0 \AA \hspace {2mm}to \hspace {2mm} 2.8 \AA \hspace {2mm} or \hspace {2mm}2.7 \AA$ is predicted to lead to a sizable enhancement of the intensity at 220 nm, with the effect being most pronounced for \alpha helices and less dramatic for 3_1_0 and \pi helices. These calculations also reveal a dependence of[\theta]_2_2_0_{nm} on {N\cdot\cdot\cdotO} separations in the range $3.0-3.5 \AA,$ a factor which may contribute to the variations in band intensities in model 3_1_0 and $\alpha$ helical peptides. With the exception ofthe Dang and Hirst proposal no testable explanations have been advanced for the observed variation in the 220 nm CD band intensity

Probing the Role of the C-H···O Hydrogen Bond Stabilized Polypeptide Chain Reversal at the C-terminus of Designed Peptide Helices. Structural Characterization of Three Decapeptides

The structural characterization in crystals of three designed decapeptides containing a double D-segment at the C-terminus is described. The crystal structures of the peptides $Boc-Leu-Aib-Val-Xxx-Leu-Aib$ $-Val-^D Ala-^DLeu-Aib-OMe$, (Xxx = Gly 2, $^DAla 3$, Aib 4) have been determined and compared with those reported earlier for peptide 1 (Xxx = Ala) and the all L analogue Boc-Leu-Aib-Val-Ala-Leu-Aib-Val-Ala-Leu-Aib-OMe, which yielded a perfect right-handed \alpha-helical structure. Peptides 1 and 2 reveal a right handed helical segment spanning residues 1 to 7, ending in a Schellman motif with $^DAla(8)$ functioning as the terminating residue. Polypeptide chain reversal occurs at residue 9, a novel feature that appears to be the consequence of a C-H···O hydrogen bond between residue 4 $C^{\alpha}H$ and residue 9 CO groups. The structures of peptides 3 and 4, which lack the pro R hydrogen at the $C^{\alpha}$ atom of residue 4, are dramatically different. Peptide 3 adopts a right-handed helical conformation over the 1 to 7 segment. Residues 8 and 9 adopt $\alpha_L$ conformations forming a C-terminus type I' \beta-turn, corresponding to an incipient left-handed twist of the polypeptide chain. In peptide 4, helix termination occurs at Aib(6), with residues 6 to 9 forming a left-handed helix, resulting in a structure that accommodates direct fusion of two helical segments of opposite twist. Peptides 3 and 4 provide examples of chiral residues occurring in the less favored sense of helical twist; $^DAla(4)$ in peptide 3 adopts an $\alpha_R$ conformation, while $^LVal(7)$ in 4 adopts an $\alpha_L$ conformation. The structural comparison of the decapeptides reported here provides evidence for the role of specific C-H···O hydrogen bonds in stabilizing chain reversals at helix termini, which may be relevant in aligning contiguous helical and strand segments in polypeptide structures

Characterization of bent helical conformations in polymorphic forms of a designed 18-residue peptide containing a central gly-pro segment

An 18-residue sequence Boc-Aib-Val-Ala-Leu-Aib-Val-Ala-Leu-Gly-Pro-Val-Ala-Leu-Aib-Val-Ala-Leu-Aib-OMe (UK18) was designed to examine the effect of introducing a Gly-Pro segment into the middle of a potentially helical peptide. The crystal structures of two polymorphic forms yielded a view of the conformation of three independent molecules. Form 1 (space group P212121, a = 14.620Å; b = 26.506Å, c = 28.858Å, Z = 4) has one molecule in the asymmetric unit, with one cocrystallized water molecule. Form 2 (space group P212121, a = 9.696Å; b = 19.641Å, c = 114.31Å, Z = 8) has two molecules in the asymmetric unit with four cocrystallized water molecules. In Form 1, residues 1 to 18 adopt φ,ψ values that lie in the right-handed helical (αR) region of the Ramachandran map. Two residues, Leu (8) (φ = -92.0°, ψ = -7.5°) and Leu (17) (φ = -94.7°, ψ = -1.7°) adopt conformations that deviate significantly from helical values. In Form 2, molecule A, residues 2 to 16 lie in the R region of φ,ψ space, with Leu (8) (φ = -94.9°, ψ = -2.9°) deviating significantly from helical values. Aib (1) and Aib (18) adopt left-handed (αL) helical conformation. Significant distortion is observed at Leu (17) (φ = -121.3°, ψ = -31.3°). Molecule B, Form 2, adopts a right-handed helix over residues 1 to 17. In all three molecules, a distinct bend in the helix is observed, with the bend angle values varying from 40.8° to 58.9°

Probing the role of the C-H center dot center dot center dot O hydrogen bond stabilized polypeptide chain reversal at the C-terminus of designed peptide helices. Structural characterization of three decapeptides

The structural characterization in crystals of three designed decapeptides containing a double D-segment at the C-terminus is described. The crystal structures of the peptides Boc-Leu-Aib-Val-Xxx-Leu-Aib-Val- (D)Ala-(D)Leu-Aib-OMe, (Xxx = Gly 2, (D)Ala 3, Aib 4) have been determined and compared with those reported earlier for peptide 1 (Xxx = Ala) and the all L analogue Boc-Leu-Aib-Val-Ala-Leu-Aib-Val-Ala-Leu-Aib-OMe, which yielded a perfect right-handed a-helical structure. Peptides 1 and 2 reveal a right-handed helical segment spanning residues 1 to 7, ending in a Schellman motif with Ala(8) functioning as the terminating residue. Polypeptide chain reversal occurs at residue 9, a novel feature that appears to be the consequence of a C-(HO)-O-... hydrogen bond between residue 4 (CH)-H-alpha and residue 9 CO groups. The structures of peptides 3 and 4, which lack the pro R hydrogen at the C-alpha atom of residue 4, are dramatically different. Peptide 3 adopts a right-handed helical conformation over the 1 to 7 segment. Residues 8 and 9 adopt at conformations forming a C-terminus type I' beta-turn, corresponding to an incipient left-handed twist of the polypeptide chain. In peptide 4, helix termination occurs at Aib(6), with residues 6 to 9 forming a left-handed helix, resulting in a structure that accommodates direct fusion of two helical segments of opposite twist. Peptides 3 and 4 provide examples of chiral residues occurring in the less favored sense of helical twist; (D)Ala(4) in peptide 3 adopts an alpha(R) conformation, while (L)Val(7) in 4 adopts an alpha(L) conformation. The structural comparison of the decapeptides reported here provides evidence for the role of specific C-(HO)-O-... hydrogen bonds in stabilizing chain reversals at helix termini, which may be relevant in aligning contiguous helical and strand segments in polypeptide structures

A right handed peptide helix containing a central double D-amino acid segment

The crystal structure of the 13 residue peptide Boc-Leu-Aib-Val-Ala-Leu-Aib-Val-DAla-DLeu-Aib-Leu-Aib-Val-OMe reveals a continuous helical conformation providing an unambiguous characterization of contiguous D-residues in a right handed peptide helix