A four stranded β-sheet structure in a designed, synthetic polypeptide

A designed four stranded β-sheet peptide has been constructed using three internal D-proline residues to nucleate β -hairpin formation

Cystine peptides: the intramolecular antiparallel β-sheet conformation of a 20-membered cyclic peptide disulfide

A 20-membered cyclic peptide disulfide has been synthesized as a conformational model for disulfide loops of limited ring size. 1H-nmr studies at 270 MHz establish the presence of three intramolecular hydrogen bonds involving the Leu, Val, and methylamide NH groups in CDCl3. Evidence for peptide aggregation in CDCl3 is also presented. A structural transition involving loosening of the hydrogen bond formed by the Val NH group is observed upon the measured addition of (CD3)2SO to CDCl3. Hydrogen-bonding studies, together with unusually low field positions of the Cys(1) and Cys(6) CαH resonances and high JHNCαH values provide support for an intramolecular antiparallel β-sheet conformation, facilitated by a chain reversal at the Aib-Ala segment. Extensive nuclear Overhauser effect studies provide compelling evidence for the proposed conformation and also establish a type I′ β -turn at the Aib-Ala residues, the site of the chain reversal

Solvent-induced β-hairpin to helix conformational transition in a designed peptide

An octapeptide containing a central Aib-Gly- segment capable of adopting β-turn conformations compatible with both hairpin (βII' or βI′) and helical (βI) structures has been designed. The effect of solvent on the conformation of the peptide Boc-Leu-Val-Val-Aib-Gly-Leu-Val-Val-OMe (VIII; Boc: t-butyloxycarbonyl; OMe: methyl ester) has been investigated by NMR and CD spectroscopy. Peptide VIII adopts a well-defined β-hairpin conformation in solvents capable of hydrogen bonding like (CD3)2SO and CD3OH. In solvents that have a lower tendency to interact with backbone peptide groups, like CDCl3 and CD3CN, helical conformations predominate. Nuclear Overhauser effects between the backbone protons and solvent shielding of NH groups involved in cross-strand hydrogen bonding, backbone chemical shifts, and vicinal coupling constants provide further support for the conformational assignments in different solvents. Truncated peptides Boc-Val-Val-Aib-Gly-Leu-Val-Val-OMe (VII), Boc-Val-Val-Aib-Gly-Leu-Val-OMe (VI), and Boc-Val-Aib-Gly-Leu-OMe (IV) were studied in CDCl3 and (CD3)2SO by 500 MHz 1H-NMR spectroscopy. Peptides IV and VI show no evidence for hairpin conformation in both the solvents. The three truncated peptides show a well-defined helical conformation in CDCl3. In (CD3)2SO, peptide VII adopts a β-hairpin conformation. The results establish that peptides may be designed, which are poised to undergo a dramatic conformational transition

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 CαH and NH resonances has been accomplished using two-dimensional correlated spectroscopy and nuclear Overhauser effects (NOEs). Analysis of interresidue NOEs and JHNCαH 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 β-turn centered at Pro(1)-Pro(2), with a cis peptide bond between these residues and a γ-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

Tryptophan-containing peptide helices: Interactions involving the indole side chain

Two designed peptide sequences containing Trp residues at positions i and i + 5 (Boc-Leu-Trp-Val-Ala-Aib-Leu-Trp-Val-OMe, 1) as well as i and i + 6 (Boc-Leu-Trp-Val-Aib-Ala-Aib-Leu-Trp-Val-OMe, 2) containing one and two centrally positioned Aib residues, respectively, for helix nucleation, have been shown to form stable helices in chloroform solutions. Structures derived from nuclear magnetic resonance (NMR) data reveal six and seven intramolecularly hydrogen-bonded NH groups in peptides 1 and 2, respectively. The helical conformation of octapeptide 1 has also been established in the solid state by X-ray diffraction. The crystal structure reveals an interesting packing motif in which helical columns are stabilized by side chain-backbone hydrogen bonding involving the indole Nε1H of Trp(2) as donor, and an acceptor C=O group from Leu(6) of a neighboring molecule. Helical columns also associate laterally, and strong interactions are observed between the Trp(2) and Trp(7) residues on neighboring molecules. The edge-to-face aromatic interactions between the indoles suggest a potential C-H…π interaction involving the Cζ3H of Trp(2). Concentration dependence of NMR chemical shifts provides evidence for peptide association in solution involving the Trp(2) Nε1H protons, presumably in a manner similar to that observed in the crystal

Peptide hairpins with strand segments containing α- and β-amino acid residues: Cross-strand aromatic interactions of facing Phe residues

The incporation of β-amino acid residues into the strand segments of designed β-hairpin leads to the formation of polar sheets, since in the case of β-peptide strands, all adjacent carbonyl groups point in one direction and the amide groups orient in the opposite direction. The conformational analysis of two designed peptide hairpins composed of α/β-hybrid segments are described: Boc-βLeu-βPhe-Val-D-Pro-Gly-βLeu-βPhe-Val-OMe (1) and Boc-βLeu-Phe-βVal-D-Pro-Gly-βLeu-Phe-βVal-OMe (2). A 500-MHz 1H-NMR (nuclear magnetic resonance) analysis in methanol supports a significant population of hairpin conformations in both peptides. Diagnostic nuclear Overhauser effects (NOEs) are observed in both cases. X-ray diffraction studies on single crystals of peptide 1 reveal a β-hairpin conformation in both the molecules, which constitute the crystallographic asymmetric unit. Three cross-strand hydrogen bonds and a nucleating type II′ β-turn at the D-Pro-Gly segment are observed in the two independent molecules. In peptide 1, the Phe residues at positions 2 and 7 occur at the nonhydrogen-bonding position, with the benzyl side chains pointing on opposite faces of the β-sheet. The observed aromatic centroid-to-centroid distances are 8.92 Å (molecule A) and 8.94 Å (molecule B). In peptide 2, the aromatic rings must occupy facing positions in antiparallel strands, in the NMR-derived structure. Peptide 1 yields a normal hairpin-like CD spectrum in methanol with a minimum at 224 nm. The CD spectrum of peptide 2 reveals a negative band at 234 nm and a positive band at 221 nm, suggestive of an exciton split doublet. Modeling of the facing Phe side chains at the hydrogen-bonding position of a canonical β-hairpin suggests that interring separation is 4.78 Å for the gauche+gauche- (g+g-) rotamer. A previously reported peptide β-hairpin composed of only α-amino acids, Boc-Leu-Phe-Val-D-Pro-Gly-Leu-Phe-Val-OMe also exhibited an anomalous far-UV (ultraviolet) CD (circular dichroism) spectrum, which was interpreted in terms of interactions between facing aromatic chromophores, Phe 2 and Phe

Genome size and ploidy influence angiosperm species' biomass under nitrogen and phosphorus limitation

Research Council of Norway. Grant Number: 196468/v40. NERC. Grant Number: NE/J012106/

Phosphate concentration and arbuscular mycorrhizal colonisation influence the growth, yield and expression of twelve PHT1 family phosphate transporters in foxtail millet (Setaria italica)

Phosphorus (P) is an essential element which plays several key roles in all living organisms. Setaria italica (foxtail millet) is a model species for panacoid grasses including several millet species widely grown in arid regions of Asia and Africa, and for the bioenergy crop switchgrass. The growth responses of S. italica to different levels of inorganic phosphate (Pi) and to colonisation with the arbuscular mycorrhizal fungus Funneliformis mosseae (syn. Glomus mosseae) were studied. Phosphate is taken up from the environment by the PHT1 family of plant phosphate transporters, which have been well characterized in several plant species. Bioinformatic analysis identified 12 members of the PHT1 gene family (SiPHT1;1-1;12) in S. italica, and RT and qPCR analysis showed that most of these transporters displayed specific expression patterns with respect to tissue, phosphate status and arbuscular mycorrhizal colonisation. SiPHT1;2 was found to be expressed in all tissues and in all growth conditions tested. In contrast, expression of SiPHT1;4 was induced in roots after 15 days growth in hydroponic medium of low Pi concentration. Expression of SiPHT1;8 and SiPHT1;9 in roots was selectively induced by colonisation with F. mosseae. SiPHT1;3 and SiPHT1;4 were found to be predominantly expressed in leaf and root tissues respectively. Several other transporters were expressed in shoots and leaves during growth in low Pi concentrations. This study will form the basis for the further characterization of these transporters, with the long term goal of improving the phosphate use efficiency of foxtail millet

Two novel hexadepsipeptides with several modified amino acid residues isolated from the fungus isaria

Two new cyclohexadepsipeptides have been isolated from the fungus Isaria. Fungal growth in solid media yielded hyphal strands from which peptide fractions were readily isolable by organic-solvent extraction. Two novel cyclodepsipeptides, isaridin A and isaridin B, have been isolated by reverse-phase HPLC, and characterized by ESI-MS and 1H-NMR. Single crystals of both peptides have been obtained, and their 3D structures were elucidated by X-ray diffraction. The isaridins contain several unusual amino acid residues. The sequences are cyclo(β-Gly-HyLeu-Pro-Phe-NMeVal-NMePhe) and cyclo(β-Gly-HyLeu-β-MePro-Phe-NMeVal-NMePhe), where NMeVal is N-methylvaline, NMePhe N-methylphenylalanine, and HyLeu hydroxyleucine (=2-hydroxy-4-methylpentanoic acid). The two peptides differ from one another at residue 3, isaridin A having an (S)-proline at this position, while β-methyl-(S)-proline (=(2S,3S)-2,3,4,5-tetrahydro-3-methyl-1H-pyrrole-2-carboxylic acid) is found in isaridin B. The solid-state conformations of both cyclic depsipeptides are characterized by the presence of two cis peptide bonds at HyLeu(2)-Pro(3)/HyLeu(2)-β-MePro(3) and NMeVal(5)-NMePhe(6), respectively. In isaridin A, a strong intramolecular H-bond is observed between Phe(4)CO...HNβ-Gly(1), and a similar, but weaker, interaction is observed between β-Gly(1)COHNPhe(4). In contrast, in isaridin B, only a single intramolecular H-bond is observed between β-Gly(1)CO...HNPhe(4)