Structures In Short Peptides Research Articles

Length-Dependent Transition from Extended to Folded Shapes in Short Oligomers of an Azetidine-Based α-Amino Acid: The Critical Role of NH···N H-Bonds.

Hydrogen bonds (H-bonds) are ubiquitous in peptides and proteins and are central to the stabilization of their structures. Inter-residue H-bonds between non-adjacent backbone amide NH and C=O motifs lead to the well-known secondary structures of helices, turns and sheets, but it is recognized that other H-bonding modes may be significant, including the weak intra-residue H-bond (called a C5 H-bond) that implicates the NH and C=O motifs of the same amino acid residue. Peptide model compounds that adopt stable C5 H-bonds are not readily available and the so-called 2.05-helix, formed by successive C5 H-bonds, is an elusive secondary structure. Using a combination of theoretical chemistry and spectroscopic studies in both the gas phase and solution phase, we have demonstrated that derivatives of 3-amino-1-methylazetidine-3-carboxylic acid, Aatc(Me) can form sidechain-backbone N-H···N C6γ H-bonds that accompany-and thereby stabilize-C5 H-bonds. In the capped trimer of Aatc(Me), extended C5/C6γ motifs are sufficiently robust to challenge classical 310-helix formation in solution and the fully-extended 2.05-helix conformer has been characterized in the gas phase. Concurrent H-bonding support for successive C5 motifs is a new axiom for stabilizing the extended backbone secondary structure in short peptides.

Simulations of Amyloid-Forming Peptides in the Crystal State

There still is little treatment available for amyloid diseases, despite their significant impact on individuals and the social and economic implications for society. One reason for this is that the physical nature of amyloid formation is not understood sufficiently well. Therefore, fundamental research at the molecular level remains necessary to support the development of therapeutics. A few structures of short peptides from amyloid-forming proteins have been determined. These can in principle be used as scaffolds for designing aggregation inhibitors. Attempts to this end have often used the tools of computational chemistry, in particular molecular simulation. However, few simulation studies of these peptides in the crystal state have been presented so far. Hence, to validate the capability of common force fields (AMBER19SB, CHARMM36m, and OPLS-AA/M) to yield insight into the dynamics and structural stability of amyloid peptide aggregates, we have performed molecular dynamics simulations of twelve different peptide crystals at two different temperatures. From the simulations, we evaluate the hydrogen bonding patterns, the isotropic B-factors, the change in energy, the Ramachandran plots, and the unit cell parameters and compare the results with the crystal structures. Most crystals are stable in the simulations but for all force fields there is at least one that deviates from the experimental crystal, suggesting more work is needed on these models.

Impact of Hydrogen‐Bond Surrogate Model on Helix Stabilization and Development of Protein‐Protein Interaction Inhibitors

AbstractProtein‐protein interactions (PPIs) involved in several diseases are predominantly mediated through short helical peptides. Unlike small molecules, due to the extended surface, the chemically constrained peptides with proper helical conformation can modulate the PPIs and thus contribute to the future drug developments. Several model systems have been introduced in the past to induce the helical structure in short peptides. Incorporating the unnatural constraints by various protocols has been excellent in biasing helical conformation and improving the proteolytic stability and the cell permeability of peptides. Hydrogen‐bond surrogate (HBS) is one of such well‐known protocols to mimic helical surfaces in numerous peptide sequences. Unlike ‘Stapled’ peptides, HBS‐model replaces the main‐chain i+4→i hydrogen bonding interaction with a covalent surrogate and retains the side‐chain functionality intact, which is beneficial for targeting multi‐faced PPIs. This review focuses on the chemistry behind the development of the HBS‐stabilized peptides, its biophysical characterizations, and numerous biological applications. Furthermore, this review will provide a broad overview of the HBS‐model to attract chemical biologists to target multifaced ‘difficult’ PPIs.

Applications of Designed Alpha‐Helical Metallopeptides

A variety of strategies have been employed to generate alpha‐helical peptides to mimic native protein‐protein interactions. Here, we present applications of a method that induces secondary structure in short peptides using engineered metal‐binding sites. We propose that the incorporation of metal ions to create helical peptides provides additional benefits that the more widely utilized covalent crosslinking methods lack. We have created a peptide system designed to bind ribose 5‐phosphate isomerase B (RpiB), a protein target in developing treatments for Chagas disease caused by Trypanosoma cruzi. In this system, we have compared peptide structure and stability when using our metal‐crosslinking system to hydrocarbon, lactam, and thioether staples across 1‐ or 2‐turns of an alpha‐helix. We have also measured the ability of each of these peptide constructs to bind RpiB and prevent homodimerization, which is required for parasitic activity. Further, we have studied whether metal‐bound peptides are better able to cross cellular membranes and enter the cytoplasm of mammalian cells compared to metal‐free peptides, as has been previously suggested in the literature, and deduced the impact of metal ion charge, identity, and secondary structure in enhanced permeability.

Application of the All-Hydrocarbon Stapling Technique in the Design of Membrane-Active Peptides.

The threats of drug resistance and new emerging pathogens have led to an urgent need to develop alternative treatment therapies. Recently, considerable research efforts have focused on membrane-active peptides (MAPs), a category of peptides in drug discovery with antimicrobial, anticancer, and cell penetration activities that have demonstrated their potential to be multifunctional agents. Nonetheless, natural MAPs have encountered various disadvantages, which mainly include poor bioavailability, the lack of a secondary structure in short peptides, and high production costs for long peptide sequences. Hence, an "all-hydrocarbon stapling system" has been applied to these peptides and proven to effectively stabilize the helical conformations, improving proteolytic resistance and increasing both the potency and the cell permeability. In this review, we summarized and categorized the advances made using this powerful technique in the development of stapled MAPs. Furthermore, outstanding issues and suggestions for future design within each subcategory were thoroughly discussed.

Synthetic Control of Tertiary Helical Structures in Short Peptides.

Helical secondary and tertiary motifs are commonly observed as binding epitopes in natural and engineered protein scaffolds. While several strategies have been described to constrain α-helices or reproduce their binding attributes in synthetic mimics, general strategies to mimic tertiary helical motifs remain in their infancy. We recently described a synthetic strategy to develop helical dimers ( J. Am. Chem. Soc. 2015, 137, 11618-11621). We found that replacement of an interhelical salt bridge with a covalent bond can stabilize antiparallel motifs in short sequences. Here we show that the approach can be generalized to obtain antiparallel and parallel dimers as well as trimer motifs. Helical stabilization requires judiciously designed cross-linkers as well as optimal interhelical hydrophobic packing. We anticipate that these mimics would afford new classes of modulators of biological function.

Remarkable Modulation of Self-Assembly in Short γ-Peptides by Neighboring Ions and Orthogonal H-Bonding.

Gabapentin, an antiepileptic drug, is known to form stable helical structures in short peptides. Distinctly, we report on the newly synthesized γ-analogue of gabapentin, that is, γ-gabapentin (γ-Gpn), which manifests β-sheet character at molecular and nanofibrous hydrogels at the supramolecular level. We investigated the influence of proximally immobilized cationic amino acids (lysine and arginine) on the self-assembly of backbone-expanded tripeptide motif. Interestingly, arginine was found to be superior, both physically and mechanically, over lysine in driving hydrogelation. We have concluded that intrinsic and biochemically distinct properties of the guanidinium ion of arginine (compared to ammonium ion of lysine) have contributed towards this effect. Furthermore, similar to pyroglutamyl (pGlu) modified amyloid β peptides, N-pGlu modification of our self-assembling tripeptide motif exerts a dramatic influence on aggregation and exhibits enhanced β-sheet character, accelerated self-assembly kinetics, improved optical transparency and provides higher mechanical stiffness to the peptide hydrogel.

Azepane Quaternary Amino Acids As Effective Inducers of 310 Helix Conformations

A simple method for the synthesis of an azepane quaternary amino acid in enantiopure form is described. Theoretical, NMR, and X-ray studies indicated that this azepane-derived amino acid is an effective stabilizer of 3(10) helical structures in short peptides.

Metal Clips That Induce Unstructured Pentapeptides To Be α-Helical In Water

Short peptides corresponding to protein helices do not form thermodynamically stable helical structures in water, a solvent that strongly competes for hydrogen-bonding amides of the peptide backbone. Metalloproteins often feature metal ions coordinated to amino acids within hydrogen-bonded helical regions of protein structure, so there is a prospect of metals stabilizing or inducing helical structures in short peptides. However, this has only previously been observed in nonaqueous solvents or under strongly helix-favoring conditions in water. Here cis-[Ru(NH(3))(4)(solvent)(2)](2+) and [Pd(en)(solvent)(2)](2+) are compared in water for their capacity as metal clips to induce alpha-helicity in completely unstructured peptides as short as five residues, Ac-HARAH-NH(2) and Ac-MARAM-NH(2). More alpha-helicity was observed for the latter pentapeptide and, when chelated to ruthenium, it showed the greatest alpha-helicity yet reported for a short metallopeptide in water (approximately 80%). Helicity was clearly induced rather than stabilized, and the two methionines were 10(13)-fold more effective than two histidines in stabilizing the lower oxidation state Ru(II) over Ru(III). The study identifies key factors that influence stability of an alpha-helical turn in water, suggests metal ions as tools for peptide folding, and raises an intriguing possibility of transiently coordinated metal ions playing important roles in native folding of polypeptides in water.

Redox Regulation of Helical Structures in Short Peptides with an Intramolecular Ferrocenyl Cross-Linking Agent

We redox-regulated alpha-helicity of short peptides intramolecularly cross-linked with a ferrocenyl linker between amino acid side chains. The helical content of the cross-linked peptide was estimated to be 56% in the neutral state of the ferrocene core at 25 degrees C. The addition of an oxidant to the solution of the cross-linked peptide enhanced the helicity up to 75%. The increased helical content returned to the same level as that in the previous ferrocene state by further addition of a reductant.

Development of a Series of Cross‐Linking Agents that Effectively Stabilize α‐Helical Structures in Various Short Peptides

A series of cross-linking agents of varying rigidity and length were designed to stabilize helical structures in short peptides and were then synthesized. The sequences of the short peptides employed in this study each include two X residues (X=Dap, Dab, Orn, and Lys) at the i/i+4, i/i+7, or i/i+11 positions to provide the sites for cross-linking. These peptides were subjected to reaction with the synthesized cross-linking agents, and the helical content of the resulting cross-linked peptides were analyzed in detail by circular dichroism. For each of the peptide classes we found combinations with the cross-linking agents suitable for the construction of stable helical structures up to >95 % helicity at 5 degrees C. Our method could also be applied to biologically related sequences seen in native proteins such as Rev.

Stabilization of Folded Peptide and Protein Structures via Distance Matching with a Long, Rigid Cross-Linker

Intramolecular cross-linking is predicted to stabilize the folded state of peptides and proteins most effectively if the cross-linker provides a rigid link that is well-matched in end-to-end distance with attachment sites in the peptide or protein. We describe a thiol-reactive sulfonated alkyne-based cross-linker that is demonstrably more effective than more flexible counterparts. Exceptional stabilization of helical structure in short peptides is obtained.

Reversible Photoregulation of Helical Structures in Short Peptides under Indoor Lighting/Dark Conditions

[reaction: see text] A photochromic cross-linking agent with a spiropyran skeleton was developed for the reversible photoregulation of helical structures in short peptides. The helical contents of the cross-linked peptides could be regulated by ambient light and dark conditions at room temperature. This switching of the helical contents could be repeated several times without substantial loss of any activity.

Effective Stabilization of α‐Helical Structures in Short Peptides with Acetylenic Cross‐Linking Agents.

AbstractFor Abstract see ChemInform Abstract in Full Text.

Effective stabilisation of α-helical structures in short peptides with acetylenic cross-linking agents

The peptides cross-linked by the acetylenic cross-linking agents showed high α-helical contents, and the α-helices thus formed survived up to substantially elevated temperature.

Enhanced protein thermostability by Ala-->Aib replacement.

The introduction into peptide chains of alpha-aminoisobutyric acid (Aib) has proven to stabilize the helical structure in short peptides by restricting the available range of polypeptide backbone conformations. In order to evaluate the potential stabilizing effect of Aib at the protein level, we have studied the conformational and stability properties of Aib-containing analogs of the carboxy-terminal subdomain 255-316 of thermolysin. Previous NMR studies have shown that this disulfide-free 62-residue fragment forms a dimer in solution and that the global 3D structure of each monomer (3 alpha-helices encompassing residues 260-274, 281-295, and 301-311) is largely coincident with that of the corresponding region in the X-ray structure of intact thermolysin. The Aib analogs of fragment 255-316 were prepared by a semisynthetic approach in which the natural fragment 255-316 was coupled to synthetic analogs of peptide 303-316 using V8-protease in 50% (v/v) aqueous glycerol [De Filippis, V., and Fontana, A. (1990) Int. J. Pept. Protein Res. 35, 219-227]. The Ala residue in position 304, 309, or 312 of fragment 255-316 was replaced by Aib, leading to the singly substituted fragments Ala304Aib, Ala309Aib, and Ala312Aib. Moreover, fragment Ala304Aib/Ala309Aib with a double Ala-->Aib exchange in positions 304 and 309 was produced. Far- and near-UV circular dichroism measurements demonstrated that both secondary and tertiary structures of the natural fragment 255-316 are fully retained upon Ala-->Aib substitution(s). Thermal unfolding measurements, carried out by recording the ellipticity at 222 nm upon heating, showed that the melting temperatures (Tm) of analogs Ala304Aib and Ala309Aib were 2.2 and 5.4 degrees C higher than that of the Ala-containing natural species (Tm = 63.5 degrees C), respectively, whereas the Tm of the Ala312Aib analog was lowered by -0.6 degree C. The enhanced stability of the Ala304Aib analog can be quantitatively explained on the basis of a reduced backbone entropy of unfolding due to the restriction of the conformational space allowed to Aib in respect to Ala, while the larger stabilization observed for the Ala309Aib analog can be accounted for by both entropic and hydrophobic effects. In fact, whereas Ala304 is a surface residue, Ala309 is shielded from the solvent, and thus the enhanced stability of fragment Ala309Aib is also due to the burial of an additional -CH3 group with respect to the natural fragment. The slightly destabilizing effect of the Ala-->Aib exchange in position 312 appears to derive from unfavorable strain energy effects, since phi and psi values for Ala312 are out of the allowed angles for Aib. Of interest, the simultaneous incorporation of Aib at positions 304 and 309 leads to a significant and additive increase of +8 degrees C in Tm. The results of this study indicate that the rational incorporation of Aib into a polypeptide chain can be a general procedure to significantly stabilize proteins.

Stabilization of alpha-helical structures in short peptides via end capping.

The alpha-helix-stabilizing effect of different amino acid residues at the helical termini of short peptides in aqueous solution has been determined. Several dodecapeptides containing alanine, asparagine, aspartate, glutamine, glutamate, and serine at the amino terminus and arginine, lysine, and alanine at the carboxyl terminus were synthesized, and the alpha-helical content of each peptide was measured by using circular dichroism spectroscopy. The trend in alpha-helix-inducing ability of these amino acids was found to be as follows: aspartate > asparagine > serine > glutamate > glutamine > alanine at the amino terminus and arginine > lysine > alanine at the carboxyl terminus. Our results agree with the Presta and Rose hypothesis [Presta, L. G. & Rose, G. D. (1988) Science 240, 1632-1641] on the role of end capping in helix stabilization.