Raheleh Rezaei Araghi

Nanoparticle‐Induced Folding and Fibril Formation of Coiled‐Coil‐Based Model Peptides

Nanomedicine is a rapidly growing field that has the potential to deliver treatments for many illnesses. However, relatively little is known about the biological risks of nanoparticles. Some studies have shown that nanoparticles can have an impact on the aggregation properties of proteins, including fibril formation. Moreover, these studies also show that the capacity of nanoscale objects to induce or prevent misfolding of the proteins strongly depends on the primary structure of the protein. Herein, light is shed on the role of the peptide primary structure in directing nanoparticle-induced misfolding by means of two model peptides. The design of these peptides is based on the alpha-helical coiled-coil folding motif, but also includes features that enable them to respond to pH changes, thus allowing pH-dependent beta-sheet formation. Previous studies showed that the two peptides differ in the pH range required for beta-sheet folding. Time-dependent circular dichroism spectroscopy and transmission electron microscopy are used to characterize peptide folding and aggregate morphology in the presence of negatively charged gold nanoparticles (AuNPs). Both peptides are found to undergo nanoparticle-induced fibril formation. The determination of binding parameters by isothermal titration calorimetry further reveals that the different propensities of both peptides to form amyloid-like structures in the presence of AuNPs is primarily due to the binding stoichiometry to the AuNPs. Modification of one of the peptide sequences shows that AuNP-induced beta-sheet formation is related to the structural propensity of the primary structure and is not a generic feature of peptide sequences with a sufficiently high binding stoichiometry to the nanoparticles.

A β/γ Motif to Mimic α-Helical Turns in Proteins

The attempt to construct nature’s architecture from nonnatural building blocks has challenged scientists for many decades. One goal of this field of study is to overcome the intrinsic protease susceptibility of natural peptides as it limits their clinical use. Peptides composed of homologous amino acids, those that have additional backbone methylene units compared to the natural a-amino acids, are at present among the most widely studied biomimetic oligomers that adopt well-defined conformations (foldamers). The wide variety of specific secondary structures that can be adopted by band g-peptides becomes especially valuable for the design of higher levels of organization such as tertiary or quaternary structures. Previous efforts towards this goal employing b-amino acid building blocks led to the discovery of both homomeric and heteromeric helix bundles and helical inhibitors of protein–protein interactions; however, there are significant differences between the packing observed in these artificial quaternary assemblies and that in the corresponding natural assemblies. This phenomenon has thus far impeded the combination of both classes into compact protein-like chimeric structures. The aim of the current study was to identify extended sequences of band g-amino acids that can be incorporated into an a-helical coiled coil to produce artificial chimeric folding motifs. Such artificial motifs with their orthogonal structural elements are great candidates for incorporation into natural helical proteins. Because protein–protein interactions involving helical domains determine specificity for important biological processes such as transcriptional control, cellular differentiation, and replication, selective disruption should be an excellent strategy for drug discovery. We were inspired by previous reports in which the principle of “equal backbone atoms” was suggested. Those designs were based on either unsubstituted or conformationally constrained amino acids. In particular b/g-hybrid peptides appear to be well-suited to mimic an a-helical conformation, thus we focused on preserving the natural side chains for the purpose of accurately imitating the natural packing in order to lend stability to the assembly. The a-helical coiled coil is a well-conserved and versatile folding motif that can serve as a model for tertiary and quaternary protein structures. This motif features a canonical heptad repeat, (abcdefg)n, in which hydrophobic residues occupy the a and d positions; these side chains make up the hydrophobic core of the interhelical interface. Charged residues at e and g generally form the second molecular recognition motif by interhelical ionic interactions. One such characteristic heptad, comprising three 13-atom hydrogen-bonded turns of the helix, can be substituted by a pentad repeat of alternating band g-amino acids with retention of the helix dipole and the formation of two 13-membered helix turns. The peptide model system described here comprises a basic a-peptide “Base-pp” which has a high propensity for heterooligomerization to an a-helical coiled coil in the presence of the acidic peptide “Acid-pp” (Figure 1 A). Heterooligomerization is driven by the burial of hydrophobic surface area, primarily contributed by Leu, and is directed by electrostatic interactions between Lys and Glu residues that flank the hydrophobic core. To evaluate b/g-hybrid peptides as a-helix mimics, the two central turns of Base-pp (positions 15–21) were replaced by a pentad of alternating band g-amino acid residues in the chimera B3b2g (Figure 1 B and C). CD spectroscopy (Figure 2 A) indicates random coil and mostly unfolded conformations for B3b2g and Acid-pp, respectively, as was expected based on the design of positions e and g. In contrast, an equimolar mixture of B3b2g and Acid-pp shows significant a-helical structure formation with two well defined minima at 208 and 222 nm. Analysis of the ellipticity at 222 nm as a function of the mole fraction of B3b2g reveals a global minimum at 0.5 (inset in Figure 2 A), which corresponds to the presence of a heteromeric assembly between Acid-pp and B3b2g with 1:1 stoichiometry. Size exclusion chromatography (SEC) was performed to characterize the oligomerization states of the peptides described above. Comparison of retention times with the peptides GCN4-p1, GCN4-pII, and GCN4-pLI as investigated by Harbury et al. suggests the presence of monomeric species (64 min) for the individual peptides Acid-pp, Base-pp and B3b2g, but the formation of four-helix-bundles (57 min) in the equimolar mixtures Acid-pp/Base-pp and Acid-pp/B3b2g (Figure 2 B). Also, [a] R. Rezaei Araghi, M. Salwiczek, S. C. Wagner, S. Wieczorek, Prof. Dr. B. Koksch Institute of Chemistry and Biochemistry, Freie Universit t Berlin Takustraße 3, 14195 Berlin (Germany) Fax: (+ 49) 30-83855644 E-mail : koksch@chemie.fu-berlin.de [b] Dr. C. J ckel Laboratory of Organic Chemistry, Eidgençssische Technische Hochschule Wolfgang-Paulistrasse 10, 8093 Z rich (Switzerland) [c] Dr. H. Cçlfen, A. Vçlkel Department of Colloid Chemistry, Max-Planck-Institute of Colloids and Interfaces 14424 Potsdam (Germany) [d] Dr. C. Baldauf BioQuant, Ruprecht-Karls-Universit t Heidelberg Im Neuenheimer Feld 267, 69120 Heidelberg (Germany) [e] Dr. C. Baldauf MPG-CAS Partner Institute for Computational Biology 320 Yue Yang Road, 200031 Shanghai (P. R. China) Fax: (+ 86) 21-54920451 E-mail : carsten@picb.ac.cn Supporting information for this article is available on the WWW under http ://dx.doi.org/10.1002/cbic.200900700.

Intramolecular Charge Interactions as a Tool to Control the Coiled‐Coil‐to‐Amyloid Transformation

Under the influence of a changed environment, amyloid-forming proteins partially unfold and assemble into insoluble beta-sheet rich fibrils. Molecular-level characterization of these assembly processes has been proven to be very challenging, and for this reason several simplified model systems have been developed over recent years. Herein, we present a series of three de novo designed model peptides that adopt different conformations and aggregate morphologies depending on concentration, pH value, and ionic strength. The design strictly follows the characteristic heptad repeat of the alpha-helical coiled-coil structural motif. In all peptides, three valine residues, known to prefer the beta-sheet conformation, have been incorporated at the solvent-exposed b, c, and f positions to make the system prone to amyloid formation. Additionally, pH-controllable intramolecular electrostatic repulsions between equally charged lysine (peptide A) or glutamate (peptide B) residues were introduced along one side of the helical cylinder. The conformational behavior was monitored by circular dichroism spectroscopic analysis and thioflavin T fluorescence, and the resulting aggregates were further characterized by transmission electron microscopy. Whereas uninterrupted alpha-helical aggregates are found at neutral pH, Coulomb repulsions between lysine residues in peptide A destabilize the helical conformation at acidic pH values and trigger an assembly into amyloid-like fibrils. Peptide B features a glutamate-based switch functionality and exhibits opposite pH-dependent folding behavior. In this case, alpha-helical aggregates are found under acidic conditions, whereas amyloids are formed at neutral pH. To further validate the pH switch concept, peptide C was designed by including serine residues, thus resulting in an equal distribution of charged residues. Surprisingly, amyloid formation is observed at all pH values investigated for peptide C. The results of further investigations into the effect of different salts, however, strongly support the crucial role of intramolecular charge repulsions in the model system presented herein.

Designing helical peptide inhibitors of protein–protein interactions

Short helical peptides combine characteristics of small molecules and large proteins and provide an exciting area of opportunity in protein design. A growing number of studies report novel helical peptide inhibitors of protein-protein interactions. New techniques have been developed for peptide design and for chemically stabilizing peptides in a helical conformation, which frequently improves protease resistance and cell permeability. We summarize advances in peptide crosslinking chemistry and give examples of peptide design studies targeting coiled-coil transcription factors, Bcl-2 family proteins, MDM2/MDMX, and HIV gp41, among other targets.

Iterative optimization yields Mcl-1-targeting stapled peptides with selective cytotoxicity to Mcl-1-dependent cancer cells

Significance Myeloid cell leukemia 1 (Mcl-1) is a key cancer survival protein that functions by binding to and blocking the activity of prodeath members of the Bcl-2 family. The prosurvival functionality of Mcl-1 can be inhibited by peptides that compete with the native prodeath factors for interaction with Mcl-1. However, unmodified peptide inhibitors of Mcl-1 are ineffective in cellular assays because they cannot access the cytoplasm. In this work, chemical modification and sequence optimization of Mcl-1 binding peptides generated compounds that have favorable biophysical properties, engage Mcl-1 in a distinctive binding mode, and can enter and selectively kill cancer cells dependent on Mcl-1 for survival. This detailed proof-of-principle study demonstrates how systematic optimization can transform a lead peptide into a drug prototype suitable for diagnostic and therapeutic development. Bcl-2 family proteins regulate apoptosis, and aberrant interactions of overexpressed antiapoptotic family members such as Mcl-1 promote cell transformation, cancer survival, and resistance to chemotherapy. Discovering potent and selective Mcl-1 inhibitors that can relieve apoptotic blockades is thus a high priority for cancer research. An attractive strategy for disabling Mcl-1 involves using designer peptides to competitively engage its binding groove, mimicking the structural mechanism of action of native sensitizer BH3-only proteins. We transformed Mcl-1–binding peptides into α-helical, cell-penetrating constructs that are selectively cytotoxic to Mcl-1–dependent cancer cells. Critical to the design of effective inhibitors was our introduction of an all-hydrocarbon cross-link or “staple” that stabilizes α-helical structure, increases target binding affinity, and independently confers binding specificity for Mcl-1 over related Bcl-2 family paralogs. Two crystal structures of complexes at 1.4 Å and 1.9 Å resolution demonstrate how the hydrophobic staple induces an unanticipated structural rearrangement in Mcl-1 upon binding. Systematic sampling of staple location and iterative optimization of peptide sequence in accordance with established design principles provided peptides that target intracellular Mcl-1. This work provides proof of concept for the development of potent, selective, and cell-permeable stapled peptides for therapeutic targeting of Mcl-1 in cancer, applying a design and validation workflow applicable to a host of challenging biomedical targets.

An unusual interstrand H-bond stabilizes the heteroassembly of helical αβγ-chimeras with natural peptides.

The substitution of α-amino acids by homologated amino acids has a strong impact on the overall structure and topology of peptides, usually leading to a loss in thermal stability. Here, we report on the identification of an ideal core packing between an α-helical peptide and an αβγ-chimera via phage display. Selected peptides assemble with the chimeric sequence with thermal stabilities that are comparable to that of the parent bundle consisting purely of α-amino acids. With the help of MD simulations and mutational analysis this stability could be explained by the formation of an interhelical H-bond between the selected cysteine and a backbone carbonyl of the β/γ-segment. Gained results can be directly applied in the design of biologically relevant peptides containing β- and γ-amino acids.

β- and γ-Amino Acids at α-Helical Interfaces: Toward the Formation of Highly Stable Foldameric Coiled Coils.

Since peptides are vital for cellular and pathogenic processes, much effort has been put into the design of unnatural oligomers that mimic natural peptide structures, also referred to as foldamers. However, to enable the specific application of foldamers, a thorough characterization of their interaction profiles in native protein environments is required. We report here the application of phage display for the identification of suitable helical environments for a sequence comprising an alternating set of β- and γ-amino acids. In vitro selected sequences show that an increase in the hydrophobic surface area at the helical interface as well as the incorporation of a polar H-bond donor functionality can significantly improve interhelical interactions involving backbone-extended amino acids. Thus, our data provide insight into the principles of the rational design of foldameric inhibitors for protein-protein interactions.

Investigation of the network of preferred interactions in an artificial coiled-coil association using the peptide array technique

Summary We screened a randomized library and identified natural peptides that bound selectively to a chimeric peptide containing α-, β- and γ-amino acids. The SPOT arrays provide a means for the systematic study of the possible interaction space accessible to the αβγ-chimera. The mutational analysis reveals the dependence of the binding affinities of α-peptides to the αβγ-chimera, on the hydrophobicity and bulkiness of the side chains at the corresponding hydrophobic interface. The stability of the resulting heteroassemblies was further confirmed in solution by CD and thermal denaturation.

The protofilament architecture of a de novo designed coiled coil-based amyloidogenic peptide

Amyloid fibrils are polymers formed by proteins under specific conditions and in many cases they are related to pathogenesis, such as Parkinsons and Alzheimers diseases. Their hallmark is the presence of a β-sheet structure. High resolution structural data on these systems as well as information gathered from multiple complementary analytical techniques is needed, from both a fundamental and a pharmaceutical perspective. Here, a previously reported de novo designed, pH-switchable coiled coil-based peptide that undergoes structural transitions resulting in fibril formation under physiological conditions has been exhaustively characterized by transmission electron microscopy (TEM), cryo-TEM, atomic force microscopy (AFM), wide-angle X-ray scattering (WAXS) and solid-state NMR (ssNMR). Overall, a unique 2-dimensional carpet-like assembly composed of large coexisiting ribbon-like, tubular and funnel-like structures with a clearly resolved protofilament substructure is observed. Whereas electron microscopy and scattering data point somewhat more to a hairpin model of β-fibrils, ssNMR data obtained from samples with selectively labelled peptides are in agreement with both, hairpin structures and linear arrangements.