Subrayashastry Aravinda

Aib residues in peptaibiotics and synthetic sequences: analysis of nonhelical conformations.

The α‐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 310‐, α‐, and mixed 310/α‐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 (αL)‐ and right (αR)‐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.

Hydrophobic peptide channels and encapsulated water wires

Peptide nanotubes with filled and empty pores and close-packed structures are formed in closely related pentapeptides. Enantiomorphic sequences, Boc-(D)Pro-Aib-Xxx-Aib-Val-OMe (Xxx = Leu, 1; Val, 2; Ala, 3; Phe, 4) and Boc-Pro-Aib-(D)Xxx-Aib-(D)Val-OMe ((D)Xxx = (D)Leu, 5; (D)Val, 6; (D)Ala, 7; (D)Phe, 8), yield molecular structures with a very similar backbone conformation but varied packing patterns in crystals. Peptides 1, 2, 5, and 6 show tubular structures with the molecules self-assembling along the crystallographic six-fold axis (c-axis) and revealing a honeycomb arrangement laterally (ab plane). Two forms of entrapped water wires have been characterized in 2: 2a with d(O...O) = 2.6 A and 2b with d(O...O) = 3.5 A. The latter is observed in 6 (6a) also. A polymorphic form of 6 (6b), grown from a solution of methanol-water, was observed to crystallize in a monoclinic system as a close-packed structure. Single-file water wire arrangements encapsulated inside hydrophobic channels formed by peptide nanotubes could be established by modeling the published structures in the cases of a cyclic peptide and a dipeptide. In all the entrapped water wires, each water molecule is involved in a hydrogen bond with a previous and succeeding water molecule. The O-H group of the water not involved in any hydrogen bond does not seem to be involved in an energetically significant interaction with the nanotube interior, a general feature of the one-dimensional water wires encapsulated in hydrophobic environments. Water wires in hydrophobic channels are contrasted with the single-file arrangements in amphipathic channels formed by aquaporins.

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 beta-turn; b) D-Pro-L-Pro-L-Xxx sequences which form type II-I (betaII-I, consecutive beta-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 beta-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 beta-turn conformation is characterized in Piv-L-Pro-L-Pro-L-Phe-OMe (18), while an example of a 5-->1 hydrogen bonded alpha-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).

A Crystalline β‐Hairpin Peptide Nucleated by a Type I′ Aib‐D‐Ala β‐Turn: Evidence for Cross‐Strand Aromatic Interactions

Recent progress in the design of beta-hairpin peptides[1] and beta-sheet models has been based on the ability to nucleate reverse turns of the appropriate stereochemistry. D-Pro-Gly[2] and to a lesser extent Asn-Gly[3] segments have been shown to facilitate formation of type I’ and II’ beta-turns, which are most often found at the site of sharp polypeptide chain reversal, that is, beta-hairpins in proteins.[4, 5] The prime turns, I’ and II’, can exert differing influences on the relative twist of the antiparallel strands. The I’ turn has the sense of twist that matches the twisting of adjacent beta-strands in proteins. In contrast, the II’ turn results in a more planar arrangement with the hairpin flattening to a considerable degree.[4a,e] The D-Pro-Xxx segment can in principle adopt both II’ and I’ turn conformations as si (D-Pro) values of +30 deg. and –120 deg. are energetically favorable.[1a, 5]

Characterization of Water Wires inside Hydrophobic Tubular Peptide Structures

The crystallographic observation of a hydrophobic, empty channel (diameter approximately 5.2 A) in the peptide Boc-(D)Pro-Aib-Leu-Aib-Val-OMe, prompted the investigation of the analog Boc-(D)Pro-Aib-Val-Aib-Val-OMe in which the side chain at position 3 was shortened, resulting in the structure of a channel (diameter approximately 7.5 A) containing a one-dimensional wire of water molecules. Crystallization in the space group P6(5) facilitates formation of a pore lined entirely by hydrocarbon side chains. Two forms of the entrapped water wires, with O...O separations of 3.5 and 2.6 A, are discussed. A lone hydrogen bond between the adjacent pairs of water molecules in the wire, with no strong interactions between the second water hydrogen and the hydrophobic walls of the channel, is a feature of the one-dimensional array. The structure provides the first crystallographic characterization of a water wire in a hydrophobic channel with implications in water and proton transport in membranes and carbon nanotubes.

Aromatic interactions in model peptide β-hairpins: Ring current effects on proton chemical shifts†

Crystal structures of eight peptide β‐hairpins in the sequence Boc‐Leu‐Phe‐Val‐Xxx‐Yyy‐Leu‐Phe‐Val‐OMe revealed that the Phe(2) and Phe(7) aromatic rings are in close spacial proximity, with the centroid–centroid distance (Rcen) of 4.4–5.4 Å between the two phenyl rings. Proton NMR spectra in chloroform and methanol solution reveal a significant upfield shift of the Phe(7) Cδ,δ′H2 protons (6.65–7.04 ppm). Specific assignments of the aromatic protons have been carried out in the peptide Boc‐Leu‐Phe‐Val‐DPro‐LPro‐Leu‐Phe‐Val‐OMe (6). The anticipated ring current shifts have been estimated from the aromatic ring geometrics observed in crystals for all eight peptides. Only one of the Cδ,δ′H proton lies in the shielding zone with rapid ring flipping, resulting in averaging between the two extreme chemical shifts. An approximate estimate of the population of conformations, which resemble crystal state orientation, may be obtained. Key nuclear Overhauser effects (NOEs) between facing Phe side chains provide support for close similarity between the solid state and solution conformation. Temperature dependence of aromatic ring proton chemical shift and line widths for peptide 6 (Boc‐Leu‐Phe‐Val‐DPro‐LPro‐Leu‐Phe‐Val‐OMe) and the control peptide Boc‐Leu‐Val‐Val‐DPro‐Gly‐Leu‐Phe‐Val‐OMe establish an enhanced barrier to ring flipping when the two Phe rings are in proximity. Modeling studies suggest that small, conformational adjustment about Cαuf8ffCβ (χ1) and Cβuf8ffCγ (χ2) bonds of both the Phe residues may be required in order to permit unhindered, uncorrelated flipping of both the Phe rings. The maintenance of the specific aromatic ring orientation in organic solvents provides evidence for significant stabilizing interaction.

Structural studies of model peptides containing beta-, gamma- and delta-amino acids.

The crystal structures of five model peptides Piv-Pro-Gly-NHMe (1), Piv-Pro-betaGly-NHMe (2), Piv-Pro-betaGly-OMe (3), Piv-Pro-deltaAva-OMe (4) and Boc-Pro-gammaAbu-OH (5) are described (Piv: pivaloyl; NHMe: N-methylamide; betaGly: beta-glycine; OMe: O-methyl ester; deltaAva: delta-aminovaleric acid; gammaAbu: gamma-aminobutyric acid). A comparison of the structures of peptides 1 and 2 illustrates the dramatic consequences upon backbone homologation in short sequences. 1 adopts a type II beta-turn conformation in the solid state, while in 2, the molecule adopts an open conformation with the beta-residue being fully extended. Piv-Pro-betaGly-OMe (3), which differs from 2 by replacement of the C-terminal NH group by an O-atom, adopts an almost identical molecular conformation and packing arrangement in the solid state. In peptide 4, the observed conformation resembles that determined for 2 and 3, with the deltaAva residue being fully extended. In peptide 5, the molecule undergoes a chain reversal, revealing a beta-turn mimetic structure stabilized by a C-H...O hydrogen bond.

Conformations of Heterochiral and Homochiral Proline-Pseudoproline Segments in Peptides: Context Dependent Cis-Trans Peptide Bond Isomerization

The pseudoproline residue (ΨPro, L‐2,2‐dimethyl‐1,3‐thiazolidine‐4‐carboxylic acid) has been introduced into heterochiral diproline segments that have been previously shown to facilitate the formation of β‐hairpins, containing central two and three residue turns. NMR studies of the octapeptide Boc‐Leu‐Phe‐Val‐DPro‐ΨPro‐Leu‐Phe‐Val‐OMe (1), Boc‐Leu‐Val‐Val‐DPro‐ΨPro‐Leu‐Val‐Val‐OMe (2), and the nonapeptide sequence Boc‐Leu‐Phe‐Val‐DPro‐ΨPro‐DAla‐Leu‐Phe‐Val‐OMe (3) established well‐registered β‐hairpin structures in chloroform solution, with the almost exclusive population of the trans conformation for the peptide bond preceding the ΨPro residue. The β‐hairpin conformation of 1 is confirmed by single crystal X‐ray diffraction. Truncation of the strand length in Boc‐Val‐DPro‐ΨPro‐Leu‐OMe (4) results in an increase in the population of the cis conformer, with a cis/trans ratio of 3.65. Replacement of ΨPro in 4 by LPro in 5, results in almost exclusive population of the trans form, resulting in an incipient β‐hairpin conformation, stabilized by two intramolecular hydrogen bonds. Further truncation of the sequence gives an appreciable rise in the population of cis conformers in the tripeptide Piv‐DPro‐ΨPro‐Leu‐OMe (6). In the homochiral segment Piv‐Pro‐ΨPro‐Leu‐OMe (7) only the cis form is observed with the NMR evidence strongly supporting a type VIa β‐turn conformation, stabilized by a 4→1 hydrogen bond between the Piv (CO) and Leu (3) NH groups. The crystal structure of the analog peptide 7a (Piv‐Pro‐ΨH,CH3Pro‐Leu‐NHMe) confirms the cis peptide bond geometry for the Pro‐ΨH,CH3Pro peptide bond, resulting in a type VIa β‐turn conformation.

Crystal structures of a new polymorphic form of gabapentin monohydrate and the e and z isomers of 4-tertiarybutylgabapentin.

Gabapentin, a widely used antiepileptic drug, crystallizes in multiple polymorphic forms. A new crystal form of gabapentin monohydrate in the space group Pbca is reported and the packing arrangement compared with that of a previously reported polymorph in the space group P21/c [Ibers, J.A. (2001) Acta Crystallogr; C57:641]. Gabapentin polymorphs can also occur from a selection of one of the two distinct chair forms of the 1,1‐disubstituted cyclohexane. Crystal structures of the E and Z isomers of 4‐tert‐butylgabapentin provide models for analyzing anticipated packing modes in the conformational isomers of gabapentin. The E isomer crystallized in the space group Pca21, while the Z isomer crystallized in the space group P21/c. The crystal structure of E‐4‐tert‐butylgabapentin provides the only example of a structure in a non‐centrosymmetric space group. Crystal structures of the hydrochloride and hydrobromide salts of 4‐tert‐butyl derivatives are reported. The results suggest that for gabapentin, a large ‘polymorph‐space’ may be anticipated, in view of the multiple conformational states that are accessible to the molecule.

Analysis of designed β-hairpin peptides: molecular conformation and packing in crystals

The crystal structures of several designed peptide hairpins have been determined in order to establish features of molecular conformations and modes of aggregation in the crystals. Hairpin formation has been induced using a centrally positioned (D)Pro-Xxx segment (Xxx = (L)Pro, Aib, Ac6c, Ala; Aib = α-aminoisobutyric acid; Ac6c = 1-aminocyclohexane-1-carboxylic acid). Structures of the peptides Boc-Leu-Phe-Val-(D)Pro-(L)Pro-Leu-Phe-Val-OMe (1), Boc-Leu-Tyr-Val-(D)Pro-(L)Pro-Leu-Phe-Val-OMe (2, polymorphic forms labeled as 2a and 2b), Boc-Leu-Val-Val-(D)Pro-(L)Pro-Leu-Val-Val-OMe (3), Boc-Leu-Phe-Val-(D)Pro-Aib-Leu-Phe-Val-OMe (4, polymorphic forms labeled as 4a and 4b), Boc-Leu-Phe-Val-(D)Pro-Ac6c-Leu-Phe-Val-OMe (5) and Boc-Leu-Phe-Val-(D)Pro-Ala-Leu-Phe-Val-OMe (6) are described. All the octapeptides adopt type II β-turn nucleated hairpins, stabilized by three or four cross-strand intramolecular hydrogen bonds. The angle of twist between the two antiparallel strands lies in the range of -9.8° to -26.7°. A detailed analysis of packing motifs in peptide hairpin crystals is presented, revealing three broad modes of association: parallel packing, antiparallel packing and orthogonal packing. An attempt to correlate aggregation modes in solution with observed packing motifs in crystals has been made by indexing of crystal faces in the case of three of the peptide hairpins. The observed modes of hairpin aggregation may be of relevance in modeling multiple modes of association, which may provide insights into the structure of insoluble polypeptide aggregates.