Synthesis and characterization of cyclic peptides that are ß-helical in trifluoroethanol.

We show that three designed cyclic d,l-peptides are ß-helical in TFE-a solvent in which the archetypal ß-helical peptide, gA, is unstructured. This result represents an advance in the field of ß-helical peptide foldamers and a step toward achieving ß-helical structure under a broad range of solvent conditions. We synthesized two of the three peptides examined using an improved variant of our original CBC strategy. Here, we began with a commercially available PEG-PS composite resin prefunctionalized with the alkanesulfonamide 'SCL' linker and preloaded with glycine. Our new conditions avoided C-terminal epimerization during the CBC step and simplified purification. In addition, we present results to define the scope and limitations of our CBC strategy. These methods and observations will prove useful in designing additional cyclic ß-helical peptides for applications ranging from transmembrane ion channels to ligands for macromolecular targets. Published 2014. This article is a U.S. Government work and is in the public domain in the USA.

Residue-dependent adsorption of model oligopeptides on gold.

The adsorption to gold surfaces in aqueous solutions has been systematically evaluated for a series of model oligopeptides. The series includes GG-X-GG "host-guest" sequences, where the central X residue is one of 19 proteinogenic amino acids, and water-soluble X5 and X10 homo-oligopeptides. Irreversible adsorption on gold of GG-X-GG peptides, which lack significant secondary structure, was quantitatively analyzed by X-ray photoelectron spectroscopy (XPS). The broad range of the quasi-equilibrium surface densities measured by XPS corroborates the hypothesis that surface interactions of GG-X-GG peptides are dominated by their central X residues. The highest surface density was produced by GGCGG, followed by sequences with hydrophobic, charged, and polar central residues. Neither electrostatic nor hydrophobic interactions dominate the adsorption of GG-X-GG peptides: for charged and polar central residues, surface densities correlate with the size of the side chains but not with the sign of the charges, while for hydrophobic residues, the surface densities are uncorrelated with side-chain hydrophobicity. An intriguing result is the disparity in surface adsorption of structural isomers of Leu and Val, which exhibit a correlation between the position of the branched carbon in the side chain and the interaction of the peptide backbone with the surface. The surface density produced by the adsorption of GG-X-GG peptides overall was low; however, adsorption tended to increase as the number of X residues increased (GG-X-GG < X5 < X10), suggesting that cooperative binding is important for surface attachment of proteins that readily adsorb on inorganic surfaces. The Leu and Val isomer investigation and trends revealed by our analysis show how the methodology and results described here provide a fundamental reference for future experimental and computational studies and for rational design of peptides that exhibit predictable adsorption behaviors on a given surface.

Circular dichroism analysis of cyclic ß-helical peptides adsorbed on planar fused quartz.

Conformational changes of three cyclic ß-helical peptides upon adsorption onto planar fused-quartz substrates were detected and analyzed by far-ultraviolet (UV) circular dichroism (CD) spectroscopy. In trifluoroethanol (TFE), hydrophobic peptides, Leu ß and Val ß, form left- and right-handed helices, respectively, and water-soluble peptide WS ß forms a left-handed helix. Upon adsorption, CD spectra showed a mixture of folded and unfolded conformations for Leu ß and Val ß and predominantly unfolded conformations for WS ß. X-ray photoelectron spectroscopy (XPS) provided insight about the molecular mechanisms governing the conformational changes, revealing that ca. 40% of backbone amides in Leu ß and Val ß were interacting with the hydrophilic substrate, while only ca. 15% of the amines/amides in WS ß showed similar interactions. In their folded ß-helical conformations, Leu ß and Val ß present only hydrophobic groups to their surroundings; hydrophilic surface groups can only interact with backbone amides if the peptides change their conformation. Conversely, as a ß helix, WS ß presents hydrophilic side chains to its surroundings that could, in principle, interact with hydrophilic surface groups, with the peptide retaining its folded structure. Instead, the observed unfolded surface conformation for WS ß and the relatively small percentage of surface-bound amides (15 versus 40% for Leu ß and Val ß) suggest that hydrophilic surface groups induce unfolding. Upon this surface-induced unfolding, WS ß interacts with the surface preferentially via hydrophilic side chains rather than backbone amides. In contrast, the unfolded ß-hairpin-like form of WS ß does not irreversibly adsorb on fused quartz from water, highlighting that solvation effects can be more important than initial conformation in governing peptide adsorption. Both label-free methods demonstrated in this work are, in general, applicable to structural analysis of a broad range of biomolecules adsorbed on transparent planar substrates, the surface properties of which could be customized.

Obesogenic diets induce anxiety in rodents: A systematic review and meta-analysis.

Obesity and mood disorders have been linked in a positive feedback loop. However, due to the bidirectional relationship between obesity and mental health, it is not clear whether anxiety is correlated with or caused by consumption of obesogenic diets. Here, we present a meta-analysis on the effects of dietary manipulation on rodent behavior in the elevated plus maze and open field test, the most common tests of anxiety-like behavior in animal models. The main dataset examined effects of obesogenic diets on time spent in the open and movement around the mazes. Auxiliary datasets examined effects of caloric restriction and protein restriction. Obesogenic diets had a negative effect on the time spent in the open overall. Effects were stronger in males than females. No effect of diet on locomotion was found, indicating that increases in anxiety-like behavior are not due to altered activity levels. No consistent effect of caloric restriction was observed, whereas protein restriction was associated with an increase in the time spent in the open. We show that obesogenic diets have a causative effect on anxiety-like behaviors in rodents, and not via a change in activity. Further studies are required to determine why obesogenic diets induce these behavioral changes.

Nutrition, anxiety and hormones. Why sex differences matter in the link between obesity and behavior.

Obesity and mood disorders are two of the most serious health issues of modern times. These health conditions are often linked, with obesity acting both as a cause and consequence of anxiety and depression. Although sex differences in the relationship between obesity and mood disorders are observed in clinical populations, the relative influence of biology versus societal conditioning is unclear. In part, this is because sex effects are rarely examined in the animal models used to derive our understanding of basic biological mechanisms. Due to the perceived confounding nature of hormonal fluctuations in females, rodent studies examining nutritional effects on behavioral responses are typically restricted to males. Yet, hormones play an important role in mediating effects of diet on behavior. In this mini-review, we outline interactions between obesity, hormones and the brain to illustrate the importance of considering sex-specific effects in studies of nutritional effects on behavior. We highlight the need for a more nuanced understanding of how dietary factors influence these relationships, arguing that such knowledge will help improve clinical health outcomes in the management of both obesity and mood disorders.

Simulations of nanocylinders self-assembled from cyclic ß-tripeptides.

This paper examines the self-assembly of cyclic ß-tripeptides using density functional theory. On the basis of literature precedents, these cyclic peptides were expected to self-assemble into cylindrical structures by stacking through backbone-backbone hydrogen bonding. Our calculations show that such stacking is energetically favorable, that the association energy per cyclic peptide decreases (becomes more favorable), and that the overall macrodipole moment of the cylindrical assembly increases with the number of stacked rings, for up to eight rings. For a structure in which two peptide ring units are joined through a single side chain-side chain covalent linker, the association energy between the two rings is favorable, albeit less so than for the unlinked rings. Significantly, the association energy in the dimers is only weakly dependent on the length (above a certain minimum) and conformation of the covalent linkers. Finally, as a plausible route for controlling assembly/disassembly of nanocylinders, we show that, for a pair of rings, each bearing a single amino-functionalized side chain, protonation of the amino group results in a strongly positive (unfavorable) association energy between the two rings.

Engineering a beta-helical D,L-peptide for folding in polar media.

Beta helices--helices formed by alternating D,L-peptides and stabilized by beta-sheet hydrogen bonding--are found naturally in only a handful of highly hydrophobic peptides. This paper explores the scope of beta-helical structure by presenting the first design and biophysical characterization of a hydrophilic D,L-peptide, 1, that forms a beta helix in methanol. The design of 1 is based on the beta-hairpin/beta helix--a new supersecondary that had been characterized previously only for hydrophobic peptides in nonpolar solvents. Incorporating polar residues in 1 provided solubility in methanol, in which the peptide adopts the expected beta-hairpin/beta-helical structure, as evidenced by CD, analytical ultracentrifugation (AUC), NMR spectroscopy, and NMR-based structure calculations. Upon titration with water (at constant peptide concentration), the structure in methanol (1 m) transitions cooperatively to an extended conformation (1 w) resembling a cyclic beta-hairpin; observation of an isodichroic point in the solvent-dependent CD spectra indicates that this transition is a two-state process. In contrast, neither 1 m nor 1 w show cooperative thermal melting; instead, their structures appear intact at temperatures as high as 65 degrees C; this observation suggests that steric constraint is dominant in stabilizing these structures. Finally, the (1)H NMR C alphaH spectroscopic resonances of 1 m are downfield-shifted with respect to random-coil values, a hitherto unreported property for beta helices that appears to be a general feature of these structures. These results show for the first time that an appropriately designed beta-helical peptide can fold stably in a polar solvent; furthermore, the structural and spectroscopic data reported should prove useful in the future design and characterization of water-soluble beta helices.

Self-assembled monolayers of alkanethiols on InAs.

We describe the deposition and properties of self-assembled monolayers (SAMs) of methyl-terminated alkanethiols on InAs(001) surface. For these model hydrophobic films, we used water contact angle measurements to survey the preparation of alkanethiol monolayers from base-activated ethanolic solutions as a function of the solution and deposition parameters, including chain length of alkanethiols, deposition time, and solution temperature and pH. We then used X-ray photoelectron spectroscopy (XPS), ellipsometry, and electrochemistry to characterize the composition and structure of octadecanethiol (ODT) monolayers deposited on InAs under optimized conditions. When applied to a thoroughly degreased InAs(001) wafer surface, the basic ODT solution removes the native oxide without excessively etching the underlying InAs(001) substrate. The resulting film contains approximately one monolayer of ODT molecules, attached to the InAs surface almost exclusively via thiolate bonds to In atoms, with organic chains extended away from the surface. These ODT monolayers are stable against degradation and oxidation in air, organic solvents, and aqueous buffers. The same base-activated ODT treatment can also be used to passivate exposed InAs/AlSb quantum well (QW) devices, preserving the unique electronic properties of InAs surfaces and allowing the operation of such passivated devices as continuous flow pH-sensors.

High-performance nanomaterials formed by rigid yet extensible cyclic ß-peptide polymers.

Organisms have evolved biomaterials with an extraordinary convergence of high mechanical strength, toughness, and elasticity. In contrast, synthetic materials excel in stiffness or extensibility, and a combination of the two is necessary to exceed the performance of natural biomaterials. We bridge this materials property gap through the side-chain-to-side-chain polymerization of cyclic ß-peptide rings. Due to their strong dipole moments, the rings self-assemble into rigid nanorods, stabilized by hydrogen bonds. Displayed amines serve as functionalization sites, or, if protonated, force the polymer to adopt an unfolded conformation. This molecular design enhances the processability and extensibility of the biopolymer. Molecular dynamics simulations predict stick-slip deformations dissipate energy at large strains, thereby, yielding toughness values greater than natural silks. Moreover, the synthesis route can be adapted to alter the dimensions and displayed chemistries of nanomaterials with mechanical properties that rival nature.

Photoswitchable Hydrogen-Bonding in Self-Organized Cylindrical Peptide Systems.

The new photochromic supramolecular system 1 is based on the E→Z isomerization of an azobenzene substituted with cyclic peptides with alternating D- and L-α-amino acids. This system allows reversible switching between inter- and intramolecularly assembled cylindrical ß-sheet structures in solution as well as in thin films at the air-water interface. Moreover, the system displays the rarely observed quantitative photoinduced E→Z isomerization.

Self-Assembling Organic Nanotubes.

Hollow tubular structures of molecular dimensions perform diverse biological functions in nature. Examples include scaffolding and packaging roles played by cytoskeletal microtubules and viral coat proteins, respectively, as well as the chemical transport and screening activities of membrane channels. In the preparation of such tubular assemblies, biological systems make extensive use of self-assembling and self-organizing strategies. Owing to numerous potential applications in areas such as chemistry, biology, and materials science considerable effort has recently been devoted to preparation of artificial nanotubular structures. This article reviews design principles and the preparation of synthetic organic nanotubes, with special emphasis on noncovalent processes such as self-assembly and self-organization.

Evaluating protocols and analytical methods for peptide adsorption experiments.

This paper evaluates analytical techniques that are relevant for performing reliable quantitative analysis of peptide adsorption on surfaces. Two salient problems are addressed: determining the solution concentrations of model GG-X-GG, X5, and X10 oligopeptides (G = glycine, X = a natural amino acid), and quantitative analysis of these peptides following adsorption on surfaces. To establish a uniform methodology for measuring peptide concentrations in water across the entire GG-X-GG and X n series, three methods were assessed: UV spectroscopy of peptides having a C-terminal tyrosine, the bicinchoninic acid (BCA) protein assay, and amino acid (AA) analysis. Due to shortcomings or caveats associated with each of the different methods, none were effective at measuring concentrations across the entire range of representative model peptides. In general, reliable measurements were within 30% of the nominal concentration based on the weight of as-received lyophilized peptide. In quantitative analysis of model peptides adsorbed on surfaces, X-ray photoelectron spectroscopy (XPS) data for a series of lysine-based peptides (GGKGG, K5, and K10) on Au substrates, and for controls incubated in buffer in the absence of peptides, suggested a significant presence of aliphatic carbon species. Detailed analysis indicated that this carbonaceous contamination adsorbed from the atmosphere after the peptide deposition. The inferred adventitious nature of the observed aliphatic carbon was supported by control experiments in which substrates were sputter-cleaned by Ar(+) ions under ultra-high vacuum (UHV) then re-exposed to ambient air. In contrast to carbon contamination, no adventitious nitrogen species were detected on the controls; therefore, the relative surface densities of irreversibly-adsorbed peptides were calculated by normalizing the N/Au ratios by the average number of nitrogen atoms per residue.

Vibrational circular-dichroism spectroscopy of homologous cyclic peptides designed to fold into ß helices of opposite chirality.

Cyclic ß-helical peptides have been developed as model structured biomolecules for examining peptide adsorption and conformation on surfaces. As a key prerequisite to circular-dichroism (CD) analysis of these model peptides on surfaces, their conformations and the corresponding vibrational spectra in the 1400-1800 cm⁻¹ range were analyzed by vibrational circular-dichroism (VCD) spectroscopy in solution. The two model peptides ("ß Leu and ß Val") were examined in chloroform, where they each fold into a homogeneous well-defined antiparallel double-stranded ß-helical species, as determined previously by NMR and electronic CD spectroscopy. Because the ß-helical conformations of ß Leu and ß Val are well characterized, the VCD spectra of these peptides can be unambiguously correlated with their structures. In addition, these two ß-helical peptides differ from one another in two key respects that make them uniquely advantageous for CD analysis--first, while their backbone conformations are topologically similar, ß Leu and ß Val form helices of opposite chiralities; second, the two peptides differ in their sequences, i.e., composition of the side chains attached to the backbone. The observed VCD spectra for ß Leu and ß Val are roughly mirror images of each other, indicating that the VCD features are dominated by the chirality and conformation of the peptide backbone rather than by the peptide sequence. Accordingly, spectra similarly characteristic of peptide secondary structure can be expected for peptides designed to be structural analogs of ß Leu and ß Val while incorporating a variety of side chains for studies of surface adsorption from organic and aqueous solvents.

Cyclic peptide helices: a hybrid beta-hairpin/beta-helical supersecondary structure.

This communication describes the design and characterization of a new peptide motif that is a hybrid of beta-hairpin and beta-helical structures. This motif was supported by CD and IR spectrocopies in nonpolar organic solvents and was confirmed by NMR spectroscopy and structure calculations using NMR-derived restraints. The beta-hairpin/beta-helical motif represents a potentially attractive new structural template for a variety of possible applications in biomolecular design, such as ligands for macromolecular targets and building blocks for new protein architectures.

Membraneless vanadium redox fuel cell using laminar flow.

This paper describes the design and characterization of a small, membraneless redox fuel cell. The smallest channel dimensions of the cell were 2 mm x 50 mum or x 200 mum; the cell was fabricated in poly(dimethylsiloxane) using soft lithography. This all-vanadium fuel cell took advantage of laminar flow to obviate the need for a membrane to separate the solutions of oxidizing and reducing components.

Design of three-dimensional, millimeter-scale models for molecular folding.

This communication describes the fabrication of three-dimensional structures of organic polymers using principles of design inspired by protein folding. The structures consist of rigid polyhedral components with dimensions of a few millimeters ("microdomains"), representing alpha-helical and beta-sheet secondary structures, connected with flexible linkers representing loops or turns. These structures were fabricated from polyurethane using photolithographic and soft lithographic techniques. The surfaces of the microdomains were patterned into hydrophobic and hydrophilic regions, and a hydrophobic photocurable liquid (serving both as lubricant and adhesive) was selectively precipitated onto the hydrophobic areas. The unfolded structures were suspended in water and agitated by tumbling. Self-assembly occurred through coalescence of the thin films of hydrophobic liquid, and was caused by minimization of the free energy of the interface between the liquid adhesive and the water. The self-assembled structures were locked in place by curing the adhesive with UV light. These results demonstrate the use of concepts abstracted from the study of proteins-including attractive hydrophobic interactions, shape complementarity, and conformational constraint-in the self-assembly of complex, three-dimensional structures on the millimeter scale.

Template-directed self-assembly of 10-microm-sized hexagonal plates.

This article presents a strategy for the fabrication of ordered microstructures using concepts of design inspired by molecular self-assembly and template-directed synthesis. The self-assembling components are 4-microm-thick hexagonal metal plates having sides 10 microm in length ("hexagons"), and each template consists of a 4-microm-thick circular metal plate surrounding a central cavity, the perimeter of which is complementary in shape to the external edges of a two-dimensional, close-packed array of hexagons. The hexagons and templates (collectively, "pieces") were fabricated via standard procedures and patterned into hydrophobic and hydrophilic regions using self-assembled monolayers (SAMs). Templated self-assembly occurs in water through capillary interactions between thin films of a nonpolar liquid adhesive coating the hydrophobic faces of the pieces. The hexagons tile the cavities enclosed by the templates, and the boundaries of the cavities determine the sizes and shapes of the assemblies. Curing the adhesive with ultraviolet light furnishes mechanically stable arrays having well-defined morphologies. By allowing control over the structures of the resulting aggregates, this work represents a step toward the development of practical methods for microfabrication based on self-assembly.