Control of peptide helix sense by temperature tuning of noncovalent chiral domino effect

We have investigated temperature effect on control of a peptide helix sense through the noncovalent chiral domino effect (NCDE: Inai, Y. et al., J. Am. Chem. Soc. 2003, 125, 8151-8162). Nonapeptide (1: Inai, Y.; Komori, H. Biomacromolecules 2004, 5, 1231-1240), which alone prefers a right-handed helix, maintained a screw-sense balance or a small imbalance at room temperature in the presence of Boc-d-amino acid. Cooling of the solution induced a left-handed helix more clearly. Conversely, heating from room temperature recovered the original right-handed sense. This helix-helix transition was essentially reversible in cooling-heating cycles. An increase in the Boc-d-amino acid concentration elevated temperature for switching CD signs based on the conformational transition. A similar thermal-driven inversion of helix sense was observed for 1 at other initial concentrations, suggesting that this behavior is insensitive to some peptide aggregation. NMR study provided direct evidence for the domino-type control of helix sense, in which Boc-Leu-OH is mainly located at the N-terminal segment. In addition, a left-handed helix induced by the d-isomer was shown to participate in equilibrium with a right-handed helix, whereas the right-handed helix was predominant in the presence of l-isomer. Consequently, we here have proposed a model for controlling a peptide helix sense (or its screw-sense bias) through temperature tuning of the external chiral interaction specific to the N-terminal sequence.     

N‐(tert‐Butoxycarbonyl)‐L‐valyl‐L‐valine methyl ester: a twisted parallel β‐sheet in the crystal structure of a protected dipeptide

The title compound, C₁₆H₃₀N₂O₅, crystallizes with three molecules in the asymmetric unit, each adopting a β‐strand/polyproline II backbone conformation. The main‐chain functional groups are hydrogen bonded into tapes having the characteristics of parallel β‐sheets. Each tape has a left‐handed twist and thus forms a helix, with six peptide molecules needed to complete a full 360° rotation. A comparison of hydrogen‐bond lengths and twisting modes is made with other related structures of protected dipeptides and with a hexapeptide derived from amyloid‐β containing the Val–Val segment. Additionally, a comparison of the backbone conformation is made with that of the Val141–Val142 segment of the water channel aquaporin‐4 (AQP4).     

Conformational interplay in hybrid peptide–helical aromatic foldamer macrocycles

Macrocyclic peptides are an important class of bioactive substances. When inserting an aromatic foldamer segment in a macrocyclic peptide, the strong folding propensity of the former may influence the conformation and alter the properties of the latter. Such an insertion is relevant because some foldamer–peptide hybrids have recently been shown to be tolerated by the ribosome, prior to forming macrocycles, and can thus be produced using an in vitro translation system. We have investigated the interplay of peptide and foldamer conformations in such hybrid macrocycles. We show that foldamer helical folding always prevails and stands as a viable means to stretch, i.e. unfold, peptides in a solvent dependent manner. Conversely, the peptide systematically has a reciprocal influence and gives rise to strong foldamer helix handedness bias as well as foldamer helix stabilisation. The hybrid macrocycles also show resistance towards proteolytic degradation.

When peptides and helical aromatic foldamers are combined in a macrocycle, an interplay of their properties is observed, including helix handedness bias, helix stabilisation, peptide stretching and peptide resistance to proteolytic degradation.     

beta-Hairpins,alpha-helices,and the intermediates among the secondary structures in the energy landscape of a peptide from a distal beta-hairpin of SH3 domain

Energy landscape of a peptide, extracted from a distal beta-hairpin of src SH3 domain, in explicit water was obtained with the multicanonical molecular dynamics. A variety of beta-hairpins with various strand-strand hydrogen bonds were found in the energy landscape at 300 K. There was no energy barrier between random-coil and hairpins. Thus, the peptide conformation can easily change from the random-coil to the hairpins in the thermal fluctuations at 300 K. The landscape also included two clusters of alpha-helices, among which an energy barrier existed, and besides, these helix clusters were separated from the other conformations. Thus, the free-energy barrier exists among the helices and the other conformations. Intermediate clusters were found between the helix and the hairpin clusters. The current study showed that the isolated state of this peptide in water fluctuates among random-coil, beta-hairpin, and alpha-helix. In SH3 domain, which has a topology of mainly beta-protein, the whole-protein folding may proceed when the segment is folded in the beta-hairpin and the other parts of the protein are coupled with the beta-hairpin in an energetically or kinetically favorite way.     

Structural characterization of the complex of the Rev response element RNA with a selected peptide

INTRODUCTION: The RSG-1.2 peptide was selected for specific binding to the Rev response element RNA, as the natural Rev peptide does. The RSG-1.2 sequence has features incompatible with the helical structure of the bound Rev peptide, indicating that it must bind in a different conformation. RESULTS: The binding of the RSG-1.2 peptide to the Rev response element RNA was characterized using multinuclear, multidimensional NMR. The RSG-1.2 peptide is shown to bind with the N-terminal segment of the peptide along the major groove in an extended conformation and turn preceding a C-terminal helical segment, which crosses the RNA groove in the region widened by the presence of purine-purine base pairs. These features make the details of the bound state rather different than that of the Rev peptide which targets the same RNA sequence binding as a single helix along the groove axis. CONCLUSIONS: These studies further demonstrate the versatility of arginine-rich peptides in recognition of specific RNA elements and the lack of conserved structural features in the bound state.     

Design of Oligourea-Based Foldamers with Antibacterial and Antifungal Activities

There is an urgent need to develop new therapeutic strategies to fight the emergence of multidrug resistant bacteria. Many antimicrobial peptides (AMPs) have been identified and characterized, but clinical translation has been limited partly due to their structural instability and degradability in physiological environments. The use of unnatural backbones leading to foldamers can generate peptidomimetics with improved properties and conformational stability. We recently reported the successful design of urea-based eukaryotic cell-penetrating foldamers (CPFs). Since cell-penetrating peptides and AMPs generally share many common features, we prepared new sequences derived from CPFs by varying the distribution of histidine- and arginine-type residues at the surface of the oligourea helix, and evaluated their activity on both Gram-positive and Gram-negative bacteria as well as on fungi. In addition, we prepared and tested new amphiphilic block cofoldamers consisting of an oligourea and a peptide segment whereby polar and charged residues are located in the peptide segment and more hydrophobic residues in the oligourea segment. Several foldamer sequences were found to display potent antibacterial activities even in the presence of 50% serum. Importantly, we show that these urea-based foldamers also possess promising antifungal properties.     

Chemical Synthesis of O‐Glycosylated Human Interleukin‐2 by the Reverse Polarity Protection Strategy

The chemical synthesis of human interleukin‐2 (IL‐2) , having a core 1 sugar, by a ligation method is reported. Although IL‐2 is a globular glycoprotein, its C‐terminal region, in particular (99‐133), is extremely insoluble when synthesized by solid‐phase method. To overcome this problem, the side‐chain carboxylic acid of the Glu residues was protected by a picolyl ester, thus reversing its polarity from negative to positive. This reverse polarity protection significantly increased the isoelectric point of the peptide segment and made it positive under acidic conditions and facilitated the purification. An efficient method to prepare the prolyl peptide thioester required for the synthesis of the (28‐65) segment was also developed. These efforts resulted in the total synthesis of the glycosylated IL‐2 having full biological activity.     

Structure and folding of glucagon-like peptide-1-(7–36)-amide in aqueous trifluoroethanol studied by NMR spectroscopy

The conformational changes of free, monomeric glucagon-like peptide-1-(7–36)-amide (GLP-1) in aqueous solution with increasing concentrations of 2,2,2-trifluoroethanol (TFE) were monitored by NMR spectroscopy. It was found that GLP-1 gradually assumes a stable, single-stranded helical structure in water solution when the TFE concentration is increased from 0 to 35% (v/v). No further structural changes were observed at higher TFE concentrations. The structure of GLP-1 in 35% TFE was determined from 292 distance restraints and 44 angle restraints by distance geometry, simulating annealing and restrained energy minimization. The helical structure extends from T7 to K28, with a less well-defined region around G16 and a disordered six-residue N-terminal domain. The folding process of GLP-1 from random coil (in water) to helix (in 35% TFE) is initiated by the formation of the C-terminal segment of the helix that is extended gradually towards the N-terminus of the peptide with increasing concentration of TFE. The exchange rates of the slow exchanging amide protons indicate that the C-terminal part of the helix is more stable than the N-terminal part. Copyright © 2001 John Wiley & Sons, Ltd.     

Facile solid-phase synthesis of sulfated tyrosine-containing peptides: Part II. Total synthesis of human big gastrin-II and its C-terminal glycine-extended peptide (G34-Gly sulfate) by the solid-phase segment condensation approach

Application of the fluoren-9-ylmethoxycarbonyl (Fmoc)-based solid-phase segment condensation approach to the preparation of sulfated peptides was investigated through the synthesis of human big gastrin-II, a 34-residue sulfated tyrosine [Tyr(SO3H)]-containing peptide. Highly acid-sensitive 2-chlorotrityl resin (Clt resin) was exclusively employed as an anchor-resin for the preparation of the three peptide segments having the C-terminal Pro residue as well as of the Tyr(SO3H)-containing resin-bound segment. By using the PyBOP-mediated coupling protocol [PyBOP=benzotriazolyloxytris(pyrrolidino)phosphonium hexafluorophosphatel, we successively condensed each segment and constructed the 34-residue peptide-resin without any difficulty. The final acid treatment of the fully protected peptide-resin at low temperature (90% aqueous TFA, 0 degree C for 8 h), which can detach a Tyr(SO3H)-containing peptide from the resin and remove the protecting groups concurrently with minimum deterioration of the sulfate, afforded a crude sulfated peptide. After one-step HPLC purification, a highly homogeneous human big gastrin-II was easily obtained in 14% yield from the protected peptide-resin. The sulfate form of the C-terminal glycine-extended gastrin (G34-Gly sulfate), a posttranslational processing intermediate of gastrin-II, was also successfully prepared with the segment condensation approach (11% yield). These results demonstrated the usefulness of the segment condensation protocol for preparing large Tyr(SO3H)-containing peptides.     

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.     

Solid state crystalline and liquid crystalline structure of semifluorinated 1-bromoalkane compounds

A series of semifluorinated 1-bromoalkane (SFBA) mesogens have been synthesized and characterized to better understand their solid state crystalline and liquid crystalline structures. In the solid state, the local conformation of the fluorocarbon segments becomes disordered once the fluorocarbon chain reaches a length above eight CF units. This is evident from the pronounced decrease of molar melting enthalpy. An increasing amount of helix and helix reverse conformations and increasingly disordered packing can also be observed with each addition of a fluorocarbon segment. X-ray diffraction peaks in the small angle region can be indexed by a tilted, two dimensional layered (herring bone) structure. The crystal structure is similar to a type of plastic crystal in which the amphiphilic character is clear, because the two segments of fluorocarbon and hydrocarbon are almost immiscible. Heating of F(CF2)12(CH2)10Br leads to a transition from plastic crystal to smectic B, as revealed by time-resolved XRD and FTIR analysis. At this solid-to-liquid transition temperature, conformational analysis confirmed an onset of the CH2 gauche conformation within the hexagonal lattice, most likely due to changes occurring in the hydrocarbon segment, and a sudden increase of helix defects along the fluorocarbon segment. The disordered helix rigid-rod structure of the fluorocarbon segment and its poor compatibility with the hydrocarbon segment play an important role in the crystalline solid and liquid crystalline structures.     

Vertical and directional insertion of helical peptide into lipid bilayer membrane

A novel helical hexadecapeptide carrying a poly(ethylene glycol) (PEG) chain at the N terminal was synthesized. The N and C terminals of the compound are labeled with a fluorescein isothiocyanate (FITC) group and an N-ethylcarbazolyl group (ECz), respectively. An octapeptide carrying the same groups and a hexadecapeptide without a PEG chain were also synthesized and used as control. A mixture of the peptide and dimyristoylphosphatidylcholine was sonicated in a buffer to prepare the liposome. The orientation as well as direction of the helical segment in the lipid bilayer were analyzed by quenching experiments of the FITC and the ECz fluorescence. The results clearly indicated that the helical segment of the peptide penetrated into the lipid bilayer with vertical orientation in both the gel and liquid crystalline states of the lipid bilayer. Notably, the bulky N terminal was left behind in the outer aqueous phase of liposome, meaning that the C terminal of the peptide points to the inner aqueous phase of liposome. The insertion mode of the helical peptide into a bilayer membrane is therefore well-regulated in terms of the orientation and the directionality by designing the balance between the PEG chain and the helix length. The methodology presented here will initiate a way to construct artificial functional molecular systems that can induce vectorial transport phenomena as seen in biological systems.     

Remote Control of the Planar Chirality in Peptide‐Bound Metallomacrocycles and Dynamic‐to‐Static Planar Chirality Control Triggered by Solvent‐Induced 310‐to‐α‐Helix Transitions

The dynamic planar chirality in a peptide‐bound Ni^II‐salphen‐based macrocycle can be remotely controlled. First, a right‐handed (P)‐3₁₀‐helix is induced in the dynamic helical oligopeptides by a chiral amino acid residue far from the macrocyclic framework. The induced planar chirality remains dynamic in chloroform and acetonitrile, but is almost completely locked in fluoroalcohols as a result of the solvent‐induced transition of the peptide chains from a 3₁₀‐helix to a wider α‐helix, which freezes the rotation of the pendant peptide units around the macrocycle.     

PepPro: A Nonredundant Structure Data Set for Benchmarking Peptide–Protein Computational Docking

We present a nonredundant benchmark, coined PepPro, for testing peptide–protein docking algorithms. Currently, PepPro contains 89 nonredundant experimentally determined peptide–protein complex structures, with peptide sequence lengths ranging from 5 to 30 amino acids. The benchmark covers peptides with distinct secondary structures, including helix, partial helix, a mixture of helix and β-sheet, β-sheet formed through binding, β-sheet formed through self-folding, and coil. In addition, unbound proteins' structures are provided for 58 complexes and can be used for testing the ability of a docking algorithm handling the conformational changes of proteins during the binding process. PepPro should benefit the docking community for the development and improvement of peptide docking algorithms. The benchmark is available at http://zoulab.dalton.missouri.edu/PepPro_benchmark . © 2019 Wiley Periodicals, Inc.     

Total chemical synthesis of large CCK isoforms using a thioester segment condensation approach

Silver-ion mediated thioester segment condensation was applied to the chemical synthesis of high molecular weight isoforms of cholecystokinin (CCK). Three building blocks, a C-terminal Tyr(SO₃H)-containing segment and two partially protected thioester segments having a C-terminal Pro residue, were prepared using Fmoc-based chemistry and 2-chlorotrityl chloride (Clt) resin as a solid support. The entire peptide chain was successfully synthesized by two consecutive silver-ion mediated condensation reactions using these building blocks. A brief TFA treatment of the final condensation product gave highly homogeneous CCK-58 in a satisfactory yield. This peptide exhibited glucose-dependent insulinotropic activity at levels comparable to CCK-33. These results demonstrate the usefulness of the silver-ion mediated segment condensation approach in the preparation of large sulfated peptides.     

Conformations of different gelatins in solutions and in films an analysis of circular dichroism (CD) measurements

The structural behaviour of gelatins from different raw materials and manufacturing processes at thermal denaturation and isothermal dehydration and rehydration is investigated by CD. At both thermal denaturation and isothermal dehydration with all gelatins examined, the triple helix content decreases. Simultaneously, the appearance of cis peptide bonds is observed. At rehydration, a structural hysteresis occurs, the reconstitution of the triple helix structure being correlated with a decrease in the content of cis peptide bonds. The possibility of the formation of chain reversals upon destruction of the triple helix is discussed.     

Control of helix sense in protein-mimicking backbone by the noncovalent chiral effect

We have reviewed our previous work regarding induction or control of a peptide helix sense through chiral stimulus to the peptide chain terminus. An optically inactive 3(10)-helix designed mainly with unusual alpha-amino acid residues was commonly employed. Such an N-terminal-free peptide generates a preferred helix sense by chiral acid molecule. A helix sense pre-directed in chiral sequence is also influenced or controlled by the chiral sign of such external molecule. Here free amide groups in the 3(10)-helical N-terminus participate in the formation of a multipoint coordinated complex. The terminal asymmetry produces the noncovalent chiral domino effect (NCDE) to influence the whole helix sense. The NCDE-mediated control of helicity provides the underlying chiral nature of protein-mimicking helical backbones: notably, chiral recognition at the terminus and modulation of helical propensity through chiral stimulus. The above items from our previous reports have been outlined and reviewed together with their significance in biopolymer science and chiral chemistry.     

Probing melittin helix-coil equilibria in solutions and vesicles

Melittin is a toxic, amphipathic peptide that rearranges from a random coil in solution to a helical structure upon binding to cell membranes or lipid vesicles. We have found that mutation of the valine at position five of the peptide to a phenylalanine or 3-nitrotyrosine induces aggregation and helix formation at low concentrations (20-80 microM). Donor-acceptor distances obtained from analyses of fluorescence energy transfer kinetics experiments with the 3-nitrotyrosine mutant indicate that both coil and helix structures are present in 2 and 20 microM aqueous solutions. The helical peptide population increases upon addition of phospholipid vesicles or in high ionic strength solutions.     

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.