Design of a "new motif" with beta-amino acids and alpha-aminoxy acids: synthesis of hybrid peptides with 12/10-helix

Hybrid peptides are prepared from a C-linked carbo-beta-amino acid ester (R-beta-Caa) and an alpha-aminoxy acid (R-Ama) derived from S-lactic acid. Extensive NMR (in CDCl 3 solution), CD, and MD studies on the tetra- and hexapeptides led to identification of robust 12/10-mixed helices. The dipeptide repeat having an R-beta-Caa and an R-Ama thus provides a "new motif" to realize a 12/10-mixed helix, for the first time, in oligomers containing R-Ama. To understand the impact of side chains in the mixed helix formation, R-beta-Caa/Ama (with no substitution in Ama) and S-beta-hAla/R-Ama oligomers were investigated. NMR studies revealed the existence of 12/10-helices in these hybrid peptides, and the side chains of monomers were found to have a profound influence on their stabilities. These observations imply that the propensity of beta-amino acid to prefer a mixed 12/10-helix governs the structural behavior in these peptides. The structural consequences of the lone-pair repulsion between nitrogen and oxygen atoms result in a new and interesting structural motif which behaves like "pseudo" beta (3),beta(2)-peptides in generating 12/10-mixed helices.     

12/10- and 11/13-mixed helices in alpha/gamma- and beta/gamma-hybrid peptides containing C-linked carbo-gamma-amino acids with alternating alpha- and beta-amino acids

New classes of alpha/gamma- and beta/gamma-hybrid peptides have been synthesized with novel 12/10- and 11/13-mixed helical patterns, respectively. The alpha/gamma-peptides were derived from the dipeptide repeats with alternating arrays of l-Ala and gamma-Caa((l)) (C-linked carbo-gamma-amino acid from d-mannose), which generated a new 12/10-mixed helix, for the first time, without a beta-amino acid. The beta/gamma-peptides made from an alternating arrangement of beta-Caa((x)) (C-linked carbo-beta-amino acid) and gamma-Caa((x)) (C-linked carbo-gamma-amino acid from d-xylose), on the other hand, resulted in an unprecedented 11/13-helix. The secondary structures in these peptides have been ascertained from detailed NMR studies, and CD spectroscopy and molecular dynamics investigations provided additional support for the structures derived.     

Induction of a heterochiral helix through the covalent chiral domino effect originating in the "Schellman motif"

We report unique phenomena where the transition from a homochiral helix to a heterochiral helix occurs by increasing the chain length of the l-sequence. Peptides composed of the l-Leu sequences with different lengths and the achiral nona-sequence at the C-terminal side were used here. Conformation of their peptides in solution was investigated mainly by using CD analysis in various solvents, or additionally by IR and NMR. When the l-sequence has a sufficient length, a left-handed helicity was induced in the achiral sequence. Notably, the polymeric l-sequence produced a heterochiral helix that switches the helix sense around the boundary of the chiral/achiral sequence. Energy calculation demonstrated that a stable heterochiral helix favors a bending form, while a homochiral helix takes a relatively straight form. Such a bending form was suggested to be advantageous to solvent effects. The "Schellman motif" has been recognized as a local heterochiral structure in protein helices. We propose a nucleation model of a heterochiral helix through the covalent chiral domino effect derived from the Schellman motif. The present findings not only offer us novel design of a heterochiral helix but also support an elementary model for the origins of homochiral-heterochiral structures from primitive chiral/achiral sequences.     

A sequence and structural study of transmembrane helices

A comparison is made between the distribution of residue preferences, three dimensional nearest neighbour contacts, preferred rotamers, helix-helix crossover angles and peptide bond angles in three sets of proteins: a non-redundant set of accurately determined globular protein structures, a set of four-helix bundle structures and a set of membrane protein structures. Residue preferences for the latter two sets may reflect overall helix stabilising propensities but may also highlight differences arising out of the contrasting nature of the solvent environments in these two cases. The results bear out the expectation that there may be differences between residue type preferences in membrane proteins and in water soluble globular proteins. For example, the -branched residue types valine and isoleucine are considerably more frequently encountered in membrane helices. Likewise, glycine and proline, residue types normally associated with `helix-breaking' propensity are found to be relatively more common in membrane helices. Three dimensional nearest neighbour contacts along the helix, preferred rotamers, and peptide bond angles are very similar in the three sets of proteins as far as can be ascertained within the limits of the relatively low resolution of the membrane proteins dataset. Crossing angles for helices in the membrane protein set resemble the four helix bundle set more than the general non-redundant set, but in contrast to both sets they have smaller crossing angles consistent with the dual requirements for the helices to form a compact structure while having to span the membrane. In addition to the pairwise packing of helices we investigate their global packing and consider the question of helix supercoiling in helix bundle proteins.     

12/14/14‐Helix Formation in 2:1 α/β‐Hybrid Peptides Containing Bicyclo[2.2.2]octane Ring Constraints

The highly constrained β‐amino acid ABOC induces different types of helices in β urea and 1:1 α/β amide oligomers. The latter can adopt 11/9‐ and 18/16‐helical folds depending on the chain length in solution. Short peptides alternating proteinogenic α‐amino acids and ABOC in a 2:1 α/β repeat pattern adopted an unprecedented and stable 12/14/14‐helix. The structure was established through extensive NMR, molecular dynamics, and IR studies. While the 1:1 α‐AA/ABOC helices diverged from the canonical α‐helix, the helix formed by the 9‐mer 2:1 α/β‐peptide allowed the projection of the α‐amino acid side chains in a spatial arrangement according to the α‐helix. Such a finding constitutes an important step toward the conception of functional tools that use the ABOC residue as a potent helix inducer for biological applications.     

Exploiting Aromatic Interactions for β‐Peptide Foldamer Helix Stabilization: A Significant Design Element

Tetrameric H10/12 helix stabilization was achieved by the application of aromatic side‐chains in β‐peptide oligomers by intramolecular backbone–side chain CH–π interactions. Because of the enlarged hydrophobic surface of the oligomers, a further aim was the investigation of the self‐assembly in a polar medium for the β‐peptide H10/12 helices. NMR, ECD, and molecular modeling results indicated that the oligomers formed by cis‐[1S,2S]‐ or cis‐[1R,2R]‐1‐amino‐1,2,3,4‐tetrahydronaphthalene‐2‐carboxylic acid (ATENAC) and cis‐[1R,2S]‐ or cis‐[1S,2R]‐2‐aminocyclohex‐3‐enecarboxylic acid (ACHEC) residues promote stable H10/12 helix formation with an alternating backbone configuration even at the tetrameric chain length. These results support the view that aromatic side‐chains can be applied for helical structure stabilization. Importantly, this is the first observation of a stable H10/12 helix with tetrameric chain‐length. The hydrophobically driven self‐assembly was achieved for the helix‐forming oligomers, seen as vesicles in transmission electron microscopy images. The self‐association phenomenon, which supports the helical secondary structure of these oligomers, depends on the hydrophobic surface area, because a higher number of aromatic side‐chains yielded larger vesicles. These results serve as an essential element for the design of helices relating to the H10/12 helix. Moreover, they open up a novel area for bioactive foldamer construction, while the hydrophobic area gained through the aromatic side‐chains may yield important receptor–ligand interaction surfaces, which can provide amplified binding strength.     

New Helical Folds in α‐Peptides with Alternating Chirality

In α‐peptides, the 8/10 helix is theoretically predicted to be energetically unstable and has not been experimentally observed so far. Based on our earlier studies on ‘helical induction’ and ‘hybrid helices’, we have adopted the ‘end‐capping’ strategy to induce the 8/10 helix in α‐peptides by using short α/β‐peptides. Thus, α‐peptides containing a regular string of α‐amino acids with alternating chirality were end capped by α/β‐peptides with 11/9‐helical motifs at the termini. Extensive NMR spectroscopy studies of these peptides revealed the presence of a hitherto unknown 8/10‐helical pattern; the H‐bonds in the shorter pseudorings were rather weak. The approach of using short helical motifs to induce new mixed helices in α‐peptides could provide avenues for more versatile design strategies.     

Thermoreversible gelation strongly coupled to polymer conformational transition

Thermoreversible gelation of polymers driven by the coil-to-helix transition in chain conformation is theoretically studied. For pairwise association of single helices, there are three fundamental types of self-assemblies as a result of competition between helix growth and helix association: Type I network (random coils connected by paired short helices), Type II network (helices connected by short random coils) and pairing (pairs of long helices without branching). Two distinct phase diagrams showing sol/gel transition and coil/helix transition are derived for weak and strong association.     

Metal‐Helix Frameworks from Short Hybrid Peptide Foldamers

The potential of structured peptides has not been explored much in the design of metal‐organic frameworks (MOFs). This is partly due to the difficulties in obtaining stable secondary structures from the short α‐peptide sequences. Here we report the design, crystal conformations, coordination site dependent different silver coordinated frameworks of short α,γ‐hybrid peptide 12‐helices consisting of terminal pyridyl moieties and the utility of metal‐helix frameworks in the adsorption of CO₂. Upon silver ion coordination the 12‐helix terminated by the 3‐pyridyl derivatives adopted a 2:2 macrocyclic structure, while the 12‐helix terminated by the 4‐pyridyl derivatives displayed remarkable porous metal‐helix frameworks. Both head‐to‐tail intermolecular H‐bonds of the 12‐helix and metal ion coordination have played an important role in stabilizing the ordered metal‐helix frameworks. The studies described here open the door to design a new class of metal‐organic‐frameworks from peptide foldamers.     

Selective covalent capture of collagen triple helices with a minimal protecting group strategy

Collagens and their most characteristic structural unit, the triple helix, play many critical roles in living systems which drive interest in preparing mimics of them. However, application of collagen mimetic helices is limited by poor thermal stability, slow rates of folding and poor equilibrium between monomer and trimer. Covalent capture of the self-assembled triple helix can solve these problems while preserving the native three-dimensional structure critical for biological function. Covalent capture takes advantage of strategically placed lysine and glutamate (or aspartate) residues which form stabilizing charge–pair interactions in the supramolecular helix and can subsequently be converted to isopeptide amide bonds under folded, aqueous conditions. While covalent capture is powerful, charge paired residues are frequently found in natural sequences which must be preserved to maintain biological function. Here we describe a minimal protecting group strategy to allow selective covalent capture of specific charge paired residues which leaves other charged residues unaltered. We investigate a series of side chain protecting groups for lysine and glutamate in model peptides for their ability to be deprotected easily and in high yield while maintaining (1) the solubility of the peptides in water, (2) the self-assembly and stability of the triple helix, and (3) the ability to covalently capture unprotected charge pairs. Optimized conditions are then illustrated in peptides derived from Pulmonary Surfactant protein A (SP-A). These covalently captured SP-A triple helices are found to have dramatically improved rates of folding and thermal stability while maintaining unmodified lysine–glutamate pairs in addition to other unmodified chemical functionality. The approach we illustrate allows for the covalent capture of collagen-like triple helices with virtually any sequence, composition or register. This dramatically broadens the utility of the covalent capture approach to the stabilization of biomimetic triple helices and thus also improves the utility of biomimetic collagens generally.

A minimal protecting group strategy is developed to allow selective covalent capture of collagen-like triple helices. This allows stabilization of this critical fold while preserving charge–pair interactions critical for biological applications.     

Structural effects on the formation of proton and alkali metal ion adducts of apolar,neutral peptides: electrospray ionization mass spectrometry and Ab initio theoretical studies

Apolar, neutral peptides have been shown to ionize extremely well under the conditions used for electrospray ionization mass spectrometry (ESIMS). Peptides for which the conformations have been independently determined in solution and in crystals have been examined by ESIMS. Studies of peptide helices ranging from 7 to 18 residues reveal that shorter helices yield exclusively singly charged ions, while in larger helices multiply charged species are detectable. Multiple sites for protonation/metallation are introduced in the helix by proline insertion or by changing the chirality in the residue. The preferred site of cation binding to helices may be the C-terminus end, where three free CO groups are available for chelation. Ab initio and DFT calculations at several levels have been carried out for the binding of H+, Li+, Na+, and K+ to CHO-(Gly)3)-OMe. The results reveal that metallation in helices is favoured by chelation to carbonyl groups at the C-terminus, while protonation involved two carbonyl groups and thus favour a 10-membered cyclic hydrogen-bonded structure. In -strands, metallation/protonation occurs at isolated carbonyl groups. Collision induced fragmentation of hydrophobic peptides under ESI conditions reveals that helix fragmentation occurs predominantly from the C-terminus, while in -hairpins cleavage occurs simultaneously at multiple sites.     

Foldamers to Nanotubes: Influence of Amino Acid Side Chains in the Hierarchical Assembly of α,γ4‐Hybrid Peptide Helices

Supramolecular assembly of various artificially folded 12‐helical architectures composed of γ⁴‐Val, γ⁴‐Leu and γ⁴‐Phe residues is investigated. In contrast to the 12‐helices composed of γ⁴‐Val and γ⁴‐Leu residues, the helices with γ⁴‐Phe residues displayed unique elongated nanotubular architectures. The elongated nanotube assembly was further explored as a template for biomineralization of silver ions to silver nanowires. A comparative study using an analogous α‐peptide helix reveals the importance of the spatial arrangement of aromatic side chains along the helical cylinder in a 12‐helix. These results suggested that the proteolytically and structurally stable α,γ⁴‐hybrid peptide 12‐helices may serve as a new generation of potential templates in the design of functional biomaterials.     

Anion-coordination-driven single–double helix switching and chiroptical molecular switching based on oligoureas

Synthetic foldamers with helical conformation are widely seen, but controllable interconversion amongst different geometries (helical structure and sense) is challenging. Here, a family of oligourea (tetra-, penta-, and hexa-) ligands bearing stereocenters at both ends are designed and shown to switch between single and double helices with concomitant inversion of helical senses upon anion coordination. The tetraurea ligand forms a right-handed single helix upon chloride anion (Cl⁻) binding and is converted into a left-handed double helix when phosphate anion (PO₄³⁻) is coordinated. The helical senses of the single and double helices are opposite, and the conversion is further found to be dependent on the stoichiometry of the ligand and phosphate anion. In contrast, only a single helix is formed for the hexaurea ligand with the phosphate anion. This distinction is attributed to the fact that the characteristic phosphate anion coordination geometry is satisfied by six urea moieties with twelve H-bonds. Our study revealed unusual single–double helix interconversion accompanied by unexpected chiroptical switching of helical senses.

Two-in-one switching of single–double helical forms and helicities is demonstrated using anion-coordination-driven oligourea foldamers.     

Influence of decavanadate clusters on the rheological properties of gelatin

The influence of polyoxovanadate clusters ([H(2)V(10)O(28)](4-)) on the thermo-reversible gelation of porcine skin gelatin solution (type A, M w approximately 40 000 g.mol (-1), pH = 3.4 < isoelectric point (IEP) approximately 8) has been investigated as a function of temperature and vanadate concentration by combining rheology and microcalorimetry. This work shows that the rheological properties of the system depend on electrostatic interactions between [H(2)V(10)O(28)](4-) and positively charged gelatin chains. In a first stage, we describe the renaturation of the gelatin triple helices in the presence of decavanadate clusters. We reveal that, when gelatin chains are in coil conformation (30 degrees C < T < 50 degrees C), the inorganic clusters act as physical cross-linkers that govern the visco-elastic properties of the mixture with an exponential dependence of the (G', G') modulus with the vanadate concentration. Below 30 degrees C, we show that gelatin triple helix nucleation is slightly favored by the presence of vanadate, but above a helix concentration of 0.012 g.cm (-3), G' is fully governed by the helix concentration. During the melting process, we reveal the non-fully reversible behavior of the vanadate/gelatin rheological properties and the stabilization of gelatin triple helices due to vanadate species until 50 degrees C. This non-reversible character has also been observed in the same experimental conditions with collagen/vanadate solutions. This is the first time that such a stabilization of triple helices has been reported in the case of gelatin hydrogels chemically cross-linked or not. We propose to analyze these results by considering that triple helix aggregates should persist because of decavanadate bridging, that the nucleation of an extended triple helix network may induce a strong modification of the vanadate cross-linker distribution in the system, or both, thus promoting the formation of thermally stable vanadate/gelatin micro-gels in the dangling end of the triple helices.     

What crystals of small analogs are trying to tell us about cellulose structure

The molecular geometries from crystal structures of 23 small molecules such as cellobiose were reviewed and extrapolated to give model cellulose chains. Within a given model, all monosaccharide units and their linkages are identical so the models are regular helices. Despite fairly large ranges for the glycosidic linkage torsion angles and , 29° and 57°, respectively, there is little variation in the n and h parameters of the model helices. They are extended, with h values (the advance per residue along the helix axis) of 5.04–5.27 Å. Some models were slightly right-handed, with n values up to 2.12 residues per helix turn. Left-handed models were in the majority, and their n values were as large as –2.91. These results are consistent with known structures of cellulose and its derivatives. An exception comes from a heavily derivatized cellobiose molecule. It yields right-handed helices with n 4.5 and h 3 Å. Because one half turn of this helix reverses the direction of the chain in a compact region, the linkage geometry is a model for chain-folding. Other derivatives that are unable to form the O3O5 hydrogen bond gave left-handed helices. The puckering of the glucose rings was also surveyed. A number of rings in small molecule structures are puckered to a degree that is similar to the puckering determined for methyl cellotrioside, cellotetraose, cellulose I and cellulose II.     

Stereoelectronic and steric effects in the collagen triple helix: toward a code for strand association

Collagen is the most abundant protein in animals. The protein consists of a helix of three strands, each with sequence X-Y-Gly. Natural collagen is most stable when X is (2S)-proline (Pro) and Y is (2S,4R)-4-hydroxyproline (4R-Hyp). We had shown previously that triple helices in which X is (2S,4S)-4-fluoroproline (4S-Flp) or Y is (2S,4R)-4-fluoroproline (4R-Flp) display hyperstability. This hyperstability arises from stereoelectronic effects that preorganize the main-chain dihedral angles in the conformation found in the triple helix. Here, we report the synthesis of strands containing both 4S-Flp in the X-position and 4R-Flp in the Y-position. We find that these strands do not form a stable triple helix, presumably because of an unfavorable steric interaction between fluoro groups on adjacent strands. Density functional theory calculations indicate that (2S,3S)-3-fluoroproline (3S-Flp), like 4S-Flp, should preorganize the main chain properly for triple-helix formation but without a steric conflict. Synthetic strands containing 3S-Flp in the X-position and 4R-Flp in the Y-position do form a triple helix. This helix is, however, less stable than one with Pro in the X-position, presumably because of an unfavorable inductive effect that diminishes the strength of the interstrand 3S-FlpC=O...H-NGly hydrogen bond. Thus, other forces can counter the benefits derived from the proper preorganization. Although (Pro-Pro-Gly)7 and (4S-Flp-4R-Flp-Gly)7 do not form stable homotrimeric helices, mixtures of these two peptides form stable heterotrimeric helices containing one (Pro-Pro-Gly)7 strand and two (4S-Flp-4R-Flp-Gly)7 strands. This stoichiometry can be understood by considering the cross sections of the two possible heterotrimeric helices. This unexpected finding portends the development of a "code" for the self-assembly of determinate triple helices from two or three strands.     

Thermoreversible gelation strongly coupled to coil-to-helix transition of polymers

This paper theoretically studies thermoreversible gelation driven by aggregation of helices formed on the polymer chains. Two fundamentally different cases of (i) multiple association of single helices and (ii) association by multiple helices with multiplicity k (such as double helices (k=2), triple helices (k=3), etc.) are treated on the basis of different equations. The helix length distribution on a polymer chain (or assemble of chains for multiple helices) is derived as a function of polymer concentration and temperature. Theoretical calculation of the total helix content in the solution is compared with experimental data of optical rotation in iota-carrageenan solutions at different polymer concentrations. It is shown that at low temperature there is a sharp transition from network to bundle state (pair, triplet, etc.). To confirm such a network/pairing transition, we carried out Monte Carlo simulation of polymer solution in which hydrogen-bonded zipper-like cross-links are formed.     

Parallel Homochiral and Anti‐Parallel Heterochiral Hydrogen‐Bonding Interfaces in Multi‐Helical Abiotic Foldamers

A hydrogen‐bonding interface between helical aromatic oligoamide foldamers has been designed to promote the folding of a helix‐turn‐helix motif with a head‐to‐tail arrangement of two helices of opposite handedness. This design complements an earlier helix‐turn‐helix motif with a head‐to‐head arrangement of two helices of identical handedness interface. The two motifs were shown to have comparable stability and were combined in a unimolecular tetra‐helix fold constituting the largest abiotic tertiary structure to date.     

Origin of the pKa perturbation of N-terminal cysteine in alpha- and 3(10)-helices: a computational DFT study

It is well documented that helices in proteins can decrease the pKa of residues located at the N-terminus, but the real nature of this perturbation remains unclear. In the present work, the origin of the effect of 3(10)- and alpha-polyalanine helices on the pKa of an N-terminal cysteine residue is examined in gas phase as well as in aqueous solution by means of density functional theory. In a systematic study of the helix dipole, the proton affinity (PA), and the pKa of the N-terminal cysteine, in relation to both the helix length and the strength of the hydrogen bonds between the helix backbone amides and the Sgamma of the N-terminal cysteine, a direct relation between the terminal hydrogen bonds and the pKa perturbation is revealed.     

Homology modeling of the receptor binding domain of human thrombopoietin

Platelet production in blood is regulated by a lineage specific humoral factor, thrombopoietin (TPO). The amino terminal domain of TPO (TPO-N) is responsible for the signal transduction mediated by the TPO receptor, c-mpl. From the predicted length of helices we found that TPO-N belongs to the long-chain subfamily of the four-helix bundle cytokine family. We built a three dimensional model of TPO-N by a comparative homology modeling procedure. The four helices of TPO-N with an up-up-down-down topology are stabilized by a tightly packed central hydrophobic core and the extended loop AB makes an additional hydrophobic core with helices B and D outside of the four helix bundle scaffold. An interpretation of the previous site directed mutageneses results in light of the model enabled us to identify two isolated receptor binding sites. The surface made of Lys 136, Lys 138 and Lys 140 in helix D, and Pro 42 and Glu 50 in loop AB forms the first receptor binding site, while the surface of Asp 8, Arg 10 and Lys14 in helix A represents the second binding site for the sequential receptor oligomerization.