Srinivasarao Raghothama

Expanding the Peptide beta-Turn in alpha gamma Hybrid Sequences: 12 Atom Hydrogen Bonded Helical and Hairpin Turns

Hybrid peptide segments containing contiguous alpha and gamma amino acid residues can form C(12) hydrogen bonded turns which may be considered as backbone expanded analogues of C(10) (beta-turns) found in alphaalpha segments. Exploration of the regular hydrogen bonded conformations accessible for hybrid alphagamma sequences is facilitated by the use of a stereochemically constrained gamma amino acid residue gabapentin (1-aminomethylcyclohexaneacetic acid, Gpn), in which the two torsion angles about C(gamma)-C(beta) (theta(1)) and C(beta)-C(alpha) (theta(2)) are predominantly restricted to gauche conformations. The crystal structures of the octapeptides Boc-Gpn-Aib-Gpn-Aib-Gpn-Aib-Gpn-Aib-OMe (1) and Boc-Leu-Phe-Val-Aib-Gpn-Leu-Phe-Val-OMe (2) reveal two distinct conformations for the Aib-Gpn segment. Peptide 1 forms a continuous helix over the Aib(2)-Aib(6) segment, while the peptide 2 forms a beta-hairpin structure stabilized by four cross-strand hydrogen bonds with the Aib-Gpn segment forming a nonhelical C(12) turn. The robustness of the helix in peptide 1 in solution is demonstrated by NMR methods. Peptide 2 is conformationally fragile in solution with evidence of beta-hairpin conformations being obtained in methanol. Theoretical calculations permit delineation of the various C(12) hydrogen bonded structures which are energetically feasible in alphagamma and gammaalpha sequences.

Antimicrobial peptides with potential for biofilm eradication: synthesis and structure activity relationship studies of battacin peptides.

We report on the first chemical syntheses and structure-activity analyses of the cyclic lipopeptide battacin which revealed that conjugation of a shorter fatty acid, 4-methyl-hexanoic acid, and linearization of the peptide sequence improves antibacterial activity and reduces hemolysis of mouse blood cells. This surprising finding of higher potency in linear lipopeptides than their cyclic counterparts is economically beneficial. This novel lipopeptide was membrane lytic and exhibited antibiofilm activity against Pseudomonas aeruginosa, Staphylococcus aureus, and, for the first time, Pseudomonas syringe pv. actinidiae. The peptide was unstructured in aqueous buffer and dimyristoylphosphatidylcholine-polymerized diacetylene vesicles, with 12% helicity induced in 50% v/v of trifluoroethanol. Our results indicate that a well-defined secondary structure is not essential for the observed antibacterial activity of this novel lipopeptide. A truncated pentapeptide conjugated to 4-methyl hexanoic acid, having similar potency against Gram negative and Gram positive pathogens was identified through alanine scanning.

β-Hairpin nucleation by Pro-Gly β-turns. Comparison of D-Pro-Gly and L-Pro-Gly sequences in an apolar octapeptide

The solution conformation of the synthetic octapeptide Boc-Leu-Val-Val-D-Pro-Gly-Leu-Val-Val-OMe 1 and Boc-Leu-Val-Val-Pro-Gly-Leu-Val-Val-OMe 2 have been investigated in organic solvents by NMR spectroscopy. Peptide 1 adopts well-defined β-hairpin conformations in CDCl3, C6D6 and (CD3)2SO, nucleated by a D-Pro-Gly Type II′ β-turn, as demonstrated by the observation of characteristic nuclear Overhauser effects (NOEs) between backbone protons and solvent shielding of NH groups involved in cross-strand hydrogen bonding. Chemical shifts and coupling constants provide further support for the β-hairpin conformation, which is consistent with the observation of a single negative circular dichroism band at 216 nm in methanol. In peptide 2, there is no characteristic interstrand NOE observed in (CD3)2SO, while in CDCl3 pronounced aggregation results in line broadening. The observation of a low temperature coefficient for the Leu(6)NH proton favours a population of Pro-Gly Type II β-turn conformations. These results suggest that in short peptide sequences, the precise nature of the β-turn is critical for hairpin formation, with Type II′ β-turns being particularly effective.

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 CαCβ (χ1) and CβCγ (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.

Multiple Conformational States in Crystals and in Solution in \alpha\gamma Hybrid Peptides. Fragility of the

The conformational properties of foldamers generated from alphagamma hybrid peptide sequences have been probed in the model sequence Boc-Aib-Gpn-Aib-Gpn-NHMe. The choice of alpha-aminoisobutyryl (Aib) and gabapentin (Gpn) residues greatly restricts sterically accessible conformational space. This model sequence was anticipated to be a short segment of the alphagamma C12 helix, stabilized by three successive 4-->1 hydrogen bonds, corresponding to a backbone-expanded analogue of the alpha polypeptide 3(10)-helix. Unexpectedly, three distinct crystalline polymorphs were characterized in the solid state by X-ray diffraction. In one form, two successive C12 hydrogen bonds were obtained at the N-terminus, while a novel C17 hydrogen-bonded gamma alpha gamma turn was observed at the C-terminus. In the other two polymorphs, isolated C9 and C7 hydrogen-bonded turns were observed at Gpn (2) and Gpn (4). Isolated C12 and C9 turns were also crystallographically established in the peptides Boc-Aib-Gpn-Aib-OMe and Boc-Gpn-Aib-NHMe, respectively. Selective line broadening of NH resonances and the observation of medium range NH(i) <--> NH(i+2) NOEs established the presence of conformational heterogeneity for the tetrapeptide in CDCl3 solution. The NMR results are consistent with the limited population of the continuous C12 helix conformation. Lengthening of the (alphagamma) n sequences in the nonapeptides Boc-Aib-Gpn-Aib-Gpn-Aib-Gpn-Aib-Gpn-Xxx (Xxx = Aib, Leu) resulted in the observation of all of the sequential NOEs characteristic of an alphagamma C12 helix. These results establish that conformational fragility is manifested in short hybrid alphagamma sequences despite the choice of conformationally constrained residues, while stable helices are formed on chain extension.

C_{12}

The handedness or chirality of molecules, organisms, and elementary particles has been widely appreciated, vindicating Pasteur’s perceptive generalization, Luni-vers est dissymktrique. Ambidexterity is rare not only in biological organisms but also in molecules. One of the most striking chiral features in biopolymers is the twist or handedness of the helices that are frequently formed. Cylindrical helical structures formed by polypeptide chains are an important constituent of peptide and protein structure, The most widespread helical structures are the alpha-helix and 310-helix, both of which have a right-handed screw sense in proteins, determined by the L configuration of the component amino acids. Peptide sequences with alternating L and D residues occur in the membrane channel-forming I 5-residue peptide gramicidin A, resulting in a large diameter cylindrical structure formed by wrapping beta strands about the helix axis.’ Alternating L,D sequences in cyclic peptides have yielded novel tubular stack. Helical polymers with alternating left- and right-handed blocks have been studies in polyisocyanates, providing new insights into the effect of chiral biases on polymer properties. In order to develop novel synthetic structures using chiral blocks, we have investigated peptides with strong helix-forming tendencies generated so as to fuse left- and right-handed cylindrical screws in the same molecule. We describe below the structural characterization of a 14-residue hydrophobic peptide containing two heptapeptide blocks of opposite chirality.

Helix in Short Sequences

Monosubstituted γ(4)-residues (γ(4)Leu, γ(4)Ile, and γ(4)Val) form helices even in short homooligomeric sequences. C14 helix formation is established by X-ray diffraction in homooligomeric (γ)n tetra-, hexa- and decapeptide sequences demonstrating the high propensity of γ residues, with proteinogenic side chains, to adopt locally folded conformations.

Ambidextrous molecules: Cylindrical peptide structures formed by fusing left- and right-handed helices

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.

Unconstrained Homooligomeric gamma-Peptides Show High Propensity for C-14 Helix Formation

Unconstrained γ(4) amino acid residues derived by homologation of proteinogenic amino acids facilitate helical folding in hybrid (αγ)n sequences. The C12 helical conformation for the decapeptide, Boc-[Leu-γ(4)(R)Val]5-OMe, is established in crystals by X-ray diffraction. A regular C12 helix is demonstrated by NMR studies of the 18 residue peptide, Boc-[Leu-γ(4)(R)Val]9-OMe, and a designed 16 residue (αγ)n peptide, incorporating variable side chains. Unconstrained (αγ)n peptides show an unexpectedly high propensity for helical folding in long polypeptide sequences.

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

The stereochemically constrained γ amino acid residue gabapentin (1‐(aminomethyl)cyclohexaneacetic acid, Gpn) has been incorporated into a host α‐peptide sequence. The structure of a hybrid αγααγα peptide, Boc‐Leu‐Gpn‐Aib‐Leu‐Gpn‐Aib‐OMe in crystals reveals a continuous helical conformation stabilized by three intramolecular 4 → 1 C12 hydrogen bonds across the αγ/αγ segments and one C10 hydrogen bond across the central αα segment. This conformation corresponds to an expanded analog of the canonical all‐α polypeptide 310‐helix, with insertion of two additional backbone atoms at each γ residue. Solvent dependence of NH chemical shifts in CDCl3 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.