Effect of heterochiral inversions on the structure of a β-hairpin peptide

We study computationally a family of β-hairpin peptides with systematically introduced chiral inversions, in explicit water, and we investigate the extent to which the backbone structure is able to fold in the presence of heterochiral perturbations. In contrast to the recently investigated case of a helical peptide, we do not find a monotonic change in secondary structure content as a function of the number of L- to D-inversions. The effects of L- to D-inversions are instead found to be highly position-specific. Additionally, in contrast to the helical peptide, some inversions increase the stability of the folded peptide: in such cases, we compute an increase in β-sheet content in the aqueous solution equilibrium ensemble. However, the tertiary structures of the stable (folded) configurations for peptides for which inversions cause an increase in β-sheet content show differences from one another, as well as from the native fold of the nonchirally perturbed β-hairpin. Our results suggest that although some chiral perturbations can increase folding stability, chirally perturbed proteins may still underperform functionally, given the relationship between structure and function.     

Effects of different force fields on the structural character of α synuclein β-hairpin peptide (35–56) in aqueous environment

The hallmark of Parkinson’s disease (PD) is the intracellular protein aggregation forming Lewy Bodies (LB) and Lewy neuritis which comprise mostly of a protein, alpha synuclein (α-syn). Molecular dynamics (MD) simulation methods can augment experimental techniques to understand misfolding and aggregation pathways with atomistic resolution. The quality of MD simulations for proteins and peptides depends greatly on the accuracy of empirical force fields. The aim of this work is to investigate the effects of different force fields on the structural character of β hairpin fragment of α-syn (residues 35–56) peptide in aqueous solution. Six independent MD simulations are done in explicit solvent using, AMBER03, AMBER99SB, GROMOS96 43A1, GROMOS96 53A6, OPLS-AA, and CHARMM27 force fields with CMAP corrections. The performance of each force field is assessed from several structural parameters such as root mean square deviation (RMSD), root mean square fluctuation (RMSF), radius of gyration (Rg), solvent accessible surface area (SASA), formation of β-turn, the stability of folded β-hairpin structure, and the favourable conformations obtained for different force fields. In this study, CMAP correction of CHARMM27 force field is found to overestimate the helical conformation, while GROMOS96 53A6 is found to most successfully capture the conformational dynamics of α-syn β-hairpin fragment as elicited from NMR.     

A prominent β-hairpin structure in the winged-helix domain of RECQ1 is required for DNA unwinding and oligomer formation

RecQ helicases have attracted considerable interest in recent years due to their role in the suppression of genome instability and human diseases. These atypical helicases exert their function by resolving a number of highly specific DNA structures. The crystal structure of a truncated catalytic core of the human RECQ1 helicase (RECQ1(49-616)) shows a prominent β-hairpin, with an aromatic residue (Y564) at the tip, located in the C-terminal winged-helix domain. Here, we show that the β-hairpin is required for the DNA unwinding and Holliday junction (HJ) resolution activity of full-length RECQ1, confirming that it represents an important determinant for the distinct substrate specificity of the five human RecQ helicases. In addition, we found that the β-hairpin is required for dimer formation in RECQ1(49-616) and tetramer formation in full-length RECQ1. We confirmed the presence of stable RECQ1(49-616) dimers in solution and demonstrated that dimer formation favours DNA unwinding; even though RECQ1 monomers are still active. Tetramers are instead necessary for more specialized activities such as HJ resolution and strand annealing. Interestingly, two independent protein-protein contacts are required for tetramer formation, one involves the β-hairpin and the other the N-terminus of RECQ1, suggesting a non-hierarchical mechanism of tetramer assembly.     

Mechanism of fiber assembly: treatment of Aβ peptide aggregation with a coarse-grained united-residue force field

The growth mechanism of β-amyloid (Aβ) peptide fibrils was studied by a physics-based coarse-grained united-residue model and molecular dynamics (MD) simulations. To identify the mechanism of monomer addition to an Aβ_1-40 fibril, we placed an unstructured monomer at a distance of 20 Å from a fibril template and allowed it to interact freely with the latter. The monomer was not biased towards fibril conformation by either the force field or the MD algorithm. With the use of a coarse-grained model with replica-exchange molecular dynamics, a longer timescale was accessible, making it possible to observe how the monomers probe different binding modes during their search for the fibril conformation. Although different assembly pathways were seen, they all follow a dock-lock mechanism with two distinct locking stages, consistent with experimental data on fibril elongation. Whereas these experiments have not been able to characterize the conformations populating the different stages, we have been able to describe these different stages explicitly by following free monomers as they dock onto a fibril template and to adopt the fibril conformation (i.e., we describe fibril elongation step by step at the molecular level). During the first stage of the assembly (“docking”), the monomer tries different conformations. After docking, the monomer is locked into the fibril through two different locking stages. In the first stage, the monomer forms hydrogen bonds with the fibril template along one of the strands in a two-stranded β-hairpin; in the second stage, hydrogen bonds are formed along the second strand, locking the monomer into the fibril structure. The data reveal a free-energy barrier separating the two locking stages. The importance of hydrophobic interactions and hydrogen bonds in the stability of the Aβ fibril structure was examined by carrying out additional canonical MD simulations of oligomers with different numbers of chains (4-16 chains), with the fibril structure as the initial conformation. The data confirm that the structures are stabilized largely by hydrophobic interactions and show that intermolecular hydrogen bonds are highly stable and contribute to the stability of the oligomers as well.     

Structure of the antimicrobial β-hairpin peptide protegrin-1 in a DLPC lipid bilayer investigated by molecular dynamics simulation

All atom molecular dynamics simulations of the 18-residue β-hairpin antimicrobial peptide protegrin-1 (PG-1, RGGRLCYCRRRFCVCVGR-NH₂) in a fully hydrated dilauroylphosphatidylcholine (DLPC) lipid bilayer have been implemented. The goal of the reported work is to investigate the structure of the peptide in a membrane environment (previously solved only in solution [R.L. Fahrner, T. Dieckmann, S.S.L. Harwig, R.I. Lehrer, D. Eisenberg, J. Feigon, Solution structure of protegrin-1, a broad-spectrum antimicrobial peptide from porcine leukocytes. Chemistry and Biology, 3 (1996) 543-550]), and to delineate specific peptide-membrane interactions which are responsible for the peptide's membrane binding properties. A novel, previously unknown, “kick” shaped conformation of the peptide was detected, where a bend at the C-terminal β-strand of the peptide caused the peptide backbone at residues 16-18 to extend perpendicular to the β-hairpin plane. This bend was driven by a highly persistent hydrogen-bond between the polar peptide side-chain of TYR7 and the unshielded backbone carbonyl oxygen atom of GLY17. The H-bond formation relieves the unfavorable free energy of insertion of polar groups into the hydrophobic membrane core. PG-1 was anchored to the membrane by strong electrostatic binding of the protonated N-terminus of the peptide to the lipid head group phosphate anions. The orientation of the peptide in the membrane, and its influence on bilayer structural and dynamic properties are in excellent agreement with solid state NMR measurements [S. Yamaguchi, T. Hong, A. Waring, R.I. Lehrer, M. Hong, Solid-State NMR Investigations of Peptide-Lipid Interaction and Orientation of a b-Sheet Antimicrobial Peptide, Protegrin, Biochemistry, 41 (2002) 9852-9862]. Importantly, two simulations which started from different initial orientations of the peptide converged to the same final equilibrium orientation of the peptide relative to the bilayer. The kick-shaped conformation was observed only in one of the two simulations.     

Molecular mechanism of action of β-hairpin antimicrobial peptide arenicin: oligomeric structure in dodecylphosphocholine micelles and pore formation in planar lipid bilayers

The membrane-active, cationic, β-hairpin peptide, arenicin, isolated from marine polychaeta Arenicola marina exhibits a broad spectrum of antimicrobial activity. The peptide in aqueous solution adopts the significantly twisted β-hairpin conformation without pronounced amphipathicity. To assess the mechanism of arenicin action, the spatial structure and backbone dynamics of the peptide in membrane-mimicking media and its pore-forming activity in planar lipid bilayers were studied. The spatial structure of the asymmetric arenicin dimer stabilized by parallel association of N-terminal strands of two β-hairpins was determined using triple-resonance nuclear magnetic resonance (NMR) spectroscopy in dodecylphosphocholine (DPC) micelles. Interaction of arenicin with micelles and its oligomerization significantly decreased the right-handed twist of the β-hairpin, increased its amphipathicity, and led to stabilization of the peptide backbone on a picosecond to nanosecond time scale. Relaxation enhancement induced by water-soluble (Mn(2+)) and lipid-soluble (16-doxylstearate) paramagnetic probes pointed to the dimer transmembrane arrangement. Qualitative NMR and circular dichroism study of arenicin-2 in mixed DPC/1,2-dioleoyl-sn-glycero-3-phosphoglycerol bicelles, sodium dodecyl sulfate micelles, and lipid vesicles confirmed that a similar dimeric assembly of the peptide was retained in membrane-mimicking systems containing negatively charged lipids and detergents. Arenicin-induced conductance was dependent on the lipid composition of the membrane. Arenicin low-conductivity pores were detected in the phosphatidylethanolamine-containing lipid mixture, whereas the high-conductivity pores were observed in an exclusively anionic lipid system. The measured conductivity levels agreed with the model in which arenicin antimicrobial activity was mediated by the formation of toroidal pores assembled of two, three, or four β-structural peptide dimers and lipid molecules. The structural transitions involved in arenicin membrane-disruptive action are discussed.     

The essentiality of B chain in stabilizing the structure of the A chain in β1-bungarotoxin fromBungarus multicinctus venom

The dynamic of Trp residue inΒ ₁-bungarotoxin (gb ₁-Bgt), the A chain ofΒ ₁-Bgt and phospholipase A₂ (PLA₂) was assessed by fluorescence measurement. Acrylamide quenching studies showed that the exposure degree of the Trp in PLA₂ is higher than the Trp inΒ ₁-Bgt. The Trp ofΒ ₁-Bgt had a higher accessibility for iodide, reflecting that the basic nature of the B chain might exert an attractive electrostatic force for iodide and increase the susceptibility of Trp in the A chain to iodide. Removal of the B chain ofΒ ₁-Bgt did not significantly affect the exposure degree of Trp in the A chain. Alternatively, the polarity of the environment around the Trp and the hydrophobic character of ANS and substrate binding sites in the separated A chain changed. Measurement of Trp fluorescence with increasing temperature showed that the stability of structure ofΒ ₁-Bgt was higher than those of the separated A chain and PLA₂. These results suggest that the B chain might interact with the A chain and stabilize the conformation of the A chain inΒ ₁-Bgt.     

What is the role of local landscape structure in the vegetation composition of field boundaries?

Question: How distinct is the flora of field boundaries? How does the structure of field boundaries determine the composition of vegetation? Location: Estonia, six 4 km × 4 km agricultural areas. Methods: We studied the vegetation of fields and field boundaries using 2 m × 2 m sample plots. We estimated the frequency of species in both habitat types, applied an MRPP test to analyse the vegetation composition of field boundaries with various combinations of landscape features (ditches, roads, tree and bush layers) illustrating this by DCA ordination, and used indicator species analysis to determine the characteristic species of each boundary type. Results: Ca. 45% of the flora of field boundaries comprised species found on agricultural land. Most typical species in fields — agrotolerants — were also the most common in field boundaries. The vegetation of road verges and grassy boundaries consisted mainly of disturbance‐tolerant species. Woody boundaries were characterised by shade‐tolerant and nitrophilous species. Ditch banks included species typical of moist habitats and semi‐natural grasslands. Few threatened or protected species were observed. Conclusion: The vegetation composition of field boundaries varied due to the complex effects of landscape structure around and in these boundaries. Plant species in agricultural landscapes can be classified into two broad emergent groups on the basis of their different responses to agricultural disturbances — agrotolerant species and nature‐value species. Agrotolerant species are promoted by agriculture, nature‐value species include rare weeds and habitat specialists. We suggest that high‐nature‐value species should prevail in monitoring the effects of land‐use intensification on biodiversity rather than total species richness.     

Crystal structure of the p38α MAP kinase in complex with a docking peptide from TAB1

The mitogen-activated protein kinase (MAPK) p38α is a key regulator in many cellular processes, whose activity is tightly regulated by upstream kinases, phosphatases and other regulators. Transforming growth factor-β activated kinase 1 (TAK1) is an upstream kinase in p38α signaling, and its full activation requires a specific activator, the TAK1-binding protein (TAB1). TAB1 was also shown to be an inducer of p38α’s autophosphorylation and/or a substrate driving the feedback control of p38α signaling. Here we determined the complex structure of the unphosphorylated p38α and a docking peptide of TAB1, which shows that the TAB1 peptide binds to the classical MAPK docking groove and induces long-range conformational changes on p38α. Our structural and biochemical analyses suggest that TAB1 is a reasonable substrate of p38α, yet the interaction between the docking peptide and p38α may not be sufficient to trigger trans-autophosphorylation of p38α.     

Effect of varying the 1–4 intramolecular scaling factor in atomistic simulations of long-chain N-alkanes with the OPLS-AA model

A comprehensive molecular dynamics simulation study of n-alkanes using the optimized potential for liquid simulation with all-atoms (OPLS-AA) force field at ambient condition has been performed. Our results indicate that while simulations with the OPLS-AA force field accurately predict the liquid state mass density for n-alkanes with carbon number equal or less than 10, for n-alkanes with carbon number equal or exceeding 12, the OPLS-AA force field with the standard scaling factor for the 1–4 intramolecular Van der Waals and electrostatic interaction gives rise to a quasi-crystalline structure. We found that accurate predictions of the liquid state properties are obtained by successively reducing the aforementioned scaling factor for each increase of the carbon number beyond n-dodecane. To better understand the effects of reducing the scaling factor, its influence on the torsion potential profile, and the corresponding gauche-trans conformer distribution, heat of vaporization, melting point, and self-diffusion coefficient for n-dodecane were investigated. This relatively simple procedure enables more accurate predictions of the thermo-physical properties of longer n-alkanes.

Why does land-use history facilitate non-native plant invasion? A field experiment with Celastrus orbiculatus in the southern Appalachians

Although historic land use is often implicated in non-native plant invasion of forests, little is known about how land-use legacies might actually facilitate invasion. We conducted a 2-year field seeding experiment in western North Carolina, USA, to compare germination and first-year seedling survival of Celastrus orbiculatus Thunb. in stands that had been cultivated and abandoned a century earlier and were dominated by tulip poplar (Liriodendron tulipifera L.), and in paired stands that had never been cultivated and were dominated by oaks (Quercus spp.). Experiments were conducted at five sites with paired tulip poplar and oak stands by varying litter mass (none, low, or high) and litter type (tulip poplar or oak). We also performed reciprocal soil translocations using pots seeded with C. orbiculatus. Soil moisture and temperature were measured throughout the growing season. Germination and survival were highest in the tulip poplar stands. Germination was also higher in plots with low litter mass. Seedling survival was highest in plots with low litter mass or no litter. Soil moisture was higher in tulip poplar stands and under low-mass litter. Differences in germination and survival among the potted plants were minimal, suggesting that soil type and ambient site conditions were less important than litter conditions for C. orbiculatus establishment. Our results suggest that the low litter mass and mesic soil conditions that are characteristic of tulip poplar stands may confer higher invasibility and explain the higher abundance of C. orbiculatus in areas with successional overstory communities associated with historically cultivated forests.     

Membrane-disruptive abilities of β-hairpin antimicrobial peptides correlate with conformation and activity: A P and H NMR study

The membrane interaction and solution conformation of two mutants of the β-hairpin antimicrobial peptide, protegrin-1 (PG-1), are investigated to understand the structural determinants of antimicrobial potency. One mutant, [A_6,8,13,15] PG-1, does not have the two disulfide bonds in wild-type PG-1, while the other, [Δ_4,18 G₁₀] PG-1, has only half the number of cationic residues. ³¹P solid-state NMR lineshapes of uniaxially aligned membranes indicate that the membrane disorder induced by the three peptides decreases in the order of PG-1>[Δ_4,18 G₁₀] PG-1?[A_6,8,13,15] PG-1. Solution NMR studies of the two mutant peptides indicate that [Δ_4,18 G₁₀] PG-1 preserves the β-hairpin fold of the wild-type peptide while [A_6,8,13,15] PG-1 adopts a random coil conformation. These NMR results correlate well with the known activities of these peptides. Thus, for this class of peptides, the presence of a β-hairpin fold is more essential than the number of cationic charges for antimicrobial activity. This study indicates that ³¹P NMR lineshapes of uniaxially aligned membranes are well correlated with antimicrobial activity, and can be used as a diagnostic tool to understand the peptide-lipid interactions of these antimicrobial peptides.     

What works in the field? A comparison of different interviewing methods in ethnobotany with special reference to the use of photographs

Ethnobotanists use a variety of interview techniques to collect ethnobotanical data. Drawing upon the results from a quantitative ethnobotanical study in five Yuracaré and Trinitario communities in the Bolivian Amazon, the pros and cons of the following methods are evaluated: (1) interviews in situ during transects, walk-in-the-woods, and homegarden sampling; and (2) interviews ex situ with fresh plant material, voucher specimens, or plant photographs as reference tools. Although the systematic use of plant photographs for ethnobotanical interviews is poorly documented in literature, the results show that indigenoùs participants in our study recognize significantly more plant species from photographs than from voucher specimens. It is argued that, especially in remote and isolated study sites, photographs might be advantageous over voucher specimens.     

Variability in secondary structure of the antimicrobial peptide Cateslytin in powder,solution, DPC micelles and at the air–water interface

Cateslytin (bCGA ³⁴⁴RSMRLSFRARGYGFR³⁵⁸), a five positively charged 15 amino-acid residues arginine-rich antimicrobial peptide, was synthesized using a very efficient procedure leading to high yields and to a 99% purity as determined by HPLC and mass spectrometry. Circular dichroism, polarized attenuated total reflectance fourier transformed infrared, polarization modulation infrared reflection Absorption spectroscopies and proton two-dimensional NMR revealed the flexibility of such a peptide. Whereas being mostly disordered as a dry powder or in water solution, the peptide acquires a α-helical character in the “membrane mimicking” solvent trifuoroethanol. In zwitterionic micelles of dodecylphophatidylcholine the helical character is retained but to a lesser extent, the peptide returning mainly to its disordered state. A β-sheet contribution of almost 100% is detected at the air–water interface. Such conformational plasticity is discussed regarding the antimicrobial action of Cateslytin. Presented at the joint biannual meeting of the SFB-GEIMM-GRIP, Anglet France, 14–19 October, 2006.     

Why is it better to produce coffee seedlings in full sunlight than in the shade? A morphophysiological approach

The coffee plant is native to shaded environments and its seedlings are often produced in shaded nurseries. However, some nursery managers, in an effort to improve the acclimation of seedlings to field conditions after transplantation, produce seedlings in full sun exposure. In this study, the morphological and physiological parameters of arabica coffee (Coffea arabica) seedlings produced in full sun (T1) and in shade (T2) were examined. The biomass accumulation and relative growth rate of T1 and T2 seedlings were similar. The T1 seedlings had less biomass allocation to shoots, a lower leaf mass ratio and a lower leaf area ratio; however, they had a greater net assimilation rate (rate of increase in plant mass per unit leaf area), which was associated with a greater net photosynthetic rate. There were no alterations in the concentrations of total chlorophylls or in the chlorophyll a/b ratio when comparing T1 and T2 seedlings. No indications of photoinhibition or photooxidative damage were observed in the T1 plants, which were shown to have a more robust antioxidant system than the T2 plants. Seedlings transferred from shade to full sun (T3) were not capable of utilising the incident extra light to fix CO₂. These seedlings showed a remarkable nocturnal retention of zeaxanthin and a significantly increased deepoxidation state of the xanthophyll cycle, even at predawn, but the activity of antioxidant enzymes was lower than in the T1 and T2 plants. Despite the acclimation capacity of T3 seedlings to the new light environment, they exhibited chronic photoinhibition and considerable photooxidative damage throughout the seven days following the transfer to full sun exposure. We further discuss the practical implications of producing coffee seedlings in full sunlight and under shade.     

43 Why G? Aggregation and gelation of GMP with XMP: the plot thickens

GMP alone, among the individual ribonucleotides, exhibits a reversible self-aggregation through hydrogen bonding to form tetrads that are the building blocks of higher order structures. These “G-tetrads” can further associate through π–π stacking to form chiral, columnar aggregates and, at higher monomer concentrations, lyotropic liquid crystalline phases. This alternate pathway for GMP should compete with its incorporation into oligonucleotides, which is why it is difficult to synthesize or amplify highly G-rich RNA or DNA with good efficiency in the absence of natural proteins, such as helicases, that function to unwind the strands. Given this competing pathway for GMP, we can ask if it came to be one of the four ribonucleotides in modern RNA in spite of, or because of, its unique properties. Our hypothesis is that the competition between reversible aggregation and covalent polymerization directed RNA toward sequences that were best suited to life on early earth. We find support in the observation that the same interactions that promote self-assembly of monomeric GMP also promote folding of G-rich RNA and DNA sequences to form inter- and intramolecular G-quadruplex structures. Such sequences are prevalent throughout the biological world and are thought to serve important functions related to genomic stability and gene regulation. G-quadruplex structures are also common motifs in aptamers, which are combinatorially derived DNA or RNA sequences that exhibit highly selective, high-affinity binding to molecular and macromolecular targets. An important consideration for GMP aggregation in a prebiotic RNA World scenario is the effect of other XMP on GMP self-assembly. In this talk, we will focus on the properties of solutions containing mixtures of GMP with AMP, CMP, and UMP. The results show that each nucleotide exerts a different influence on the self-assembly of GMP, raising interesting questions about scenarios on prebiotic Earth that would be consistent with abiotic RNA polymerization.     

Structural study of the β-hairpin marine antimicrobial peptide arenicin-2 in PC/PG lipid bilayers by fourier transform infrared spectroscopy

Arenicin-2 is a 21-residue β-hairpin antimicrobial peptide isolated from the marine lugworm Arenicola marina. The structure of this cationic peptide in partly charged lipid membrane made of PC/PG (7: 3) was studied by FTIR, CD, and Trp fluorescence spectroscopies. FTIR spectra of arenicin in amide I region were analyzed using curve-fitting and second derivative procedures. The FTIR data for the peptide in PC/PG liposomes were compared with the data obtained in anionic SDS micelles where arenicin forms a dimer stabilized by parallel association of two β-hairpins according to previous NMR spectroscopy studies [Ovchinnikova et al., Biopolymers, 2007, vol. 89, pp. 455–464; Shenkarev et al., Biochemistry, 2011, vol. 50, pp. 6255–6265]. The results obtained in present work indicate that arenicin forms the dimeric structure in partly charged PC/PG lipid membrane. This finding is discussed in relation to interpretation of low-conducting pores observed for arenicin in negatively charged membranes.