Early diagnosis and treatment of Alzheimer's disease by targeting toxic soluble Aß oligomers.

Transient soluble oligomers of amyloid-ß (Aß) are toxic and accumulate early prior to insoluble plaque formation and cognitive impairment in Alzheimer's disease (AD). Synthetic cyclic D,L-α-peptides (e.g., 1) self-assemble into cross ß-sheet nanotubes, react with early Aß species (1-3 mers), and inhibit Aß aggregation and toxicity in stoichiometric concentrations, in vitro. Employing a semicarbazide as an aza-glycine residue with an extra hydrogen-bond donor to tune nanotube assembly and amyloid engagement, [azaGly6]-1 inhibited Aß aggregation and toxicity at substoichiometric concentrations. High-resolution NMR studies revealed dynamic interactions between [azaGly6]-1 and Aß42 residues F19 and F20, which are pivotal for early dimerization and aggregation. In an AD mouse model, brain positron emission tomography (PET) imaging using stable 64Cu-labeled (aza)peptide tracers gave unprecedented early amyloid detection in 44-d presymptomatic animals. No tracer accumulation was detected in the cortex and hippocampus of 44-d-old 5xFAD mice; instead, intense PET signal was observed in the thalamus, from where Aß oligomers may spread to other brain parts with disease progression. Compared with standard 11C-labeled Pittsburgh compound-B (11C-PIB), which binds specifically fibrillar Aß plaques, 64Cu-labeled (aza)peptide gave superior contrast and uptake in young mouse brain correlating with Aß oligomer levels. Effectively crossing the blood-brain barrier (BBB), peptide 1 and [azaGly6]-1 reduced Aß oligomer levels, prolonged lifespan of AD transgenic Caenorhabditis elegans, and abated memory and behavioral deficits in nematode and murine AD models. Cyclic (aza)peptides offer novel promise for early AD diagnosis and therapy.

Structural insights into the role of the WW2 domain on tandem WW-PPxY motif interactions of oxidoreductase WWOX.

Class I WW domains are present in many proteins of various functions and mediate protein interactions by binding to short linear PPxY motifs. Tandem WW domains often bind peptides with multiple PPxY motifs, but the interplay of WW-peptide interactions is not always intuitive. The WW domain-containing oxidoreductase (WWOX) harbors two WW domains: an unstable WW1 capable of PPxY binding and stable WW2 that cannot bind PPxY. The WW2 domain has been suggested to act as a WW1 domain chaperone, but the underlying mechanism of its chaperone activity remains to be revealed. Here, we combined NMR, isothermal calorimetry, and structural modeling to elucidate the roles of both WW domains in WWOX binding to its PPxY-containing substrate ErbB4. Using NMR, we identified an interaction surface between these two domains that supports a WWOX conformation compatible with peptide substrate binding. Isothermal calorimetry and NMR measurements also indicated that while binding affinity to a single PPxY motif is marginally increased in the presence of WW2, affinity to a dual-motif peptide increases 10-fold. Furthermore, we found WW2 can directly bind double-motif peptides using its canonical binding site. Finally, differential binding of peptides in mutagenesis experiments was consistent with a parallel N- to C-terminal PPxY tandem motif orientation in binding to the WW1-WW2 tandem domain, validating structural models of the interaction. Taken together, our results reveal the complex nature of tandem WW-domain organization and substrate binding, highlighting the contribution of WWOX WW2 to both protein stability and target binding.

Inherent Minor Conformer of Bordetella Effector BteA Directs Chaperone-Mediated Unfolding.

The pathogen Bordetella pertussis uses a type-3 secretion system (T3SS) to inject its cytotoxic effector BteA into the host cell via a designated needle structure. Prior to injection BteA is bound to its cognate chaperone BtcA presumed to assist in effector unfolding en route to needle passage. We utilized NMR and EPR spectroscopy to uncover the molecular mechanism of BtcA-mediated unfolding of BteA. BtcA induces a global structural change in the effector, which adopts a more extended and partially unfolded conformation. EPR distance measurements further show that the structured helical-bundle form of free BteA exists in conformational equilibrium with a lowly populated minor species. The nature of this equilibrium was probed using NMR relaxation dispersion experiments. At 283 K structural effects are most pronounced for a contiguous surface spanning the A- and B-helices of BteA, extending at 303 K to a second surface including the D- and E-helices. Residues perturbed in the minor conformation coincide with those exhibiting a BtcA-induced increase in flexibility, identifying this conformation as the BtcA-bound form of the effector. Our findings hint at a conformational-selectivity mechanism for the chaperone interaction with the effector, a paradigm that may be common to effector-chaperones secretion complexes in this family of pathogens.

Investigation of a KcsA Cytoplasmic pH Gate in Lipoprotein Nanodiscs.

The bacterial potassium channel KcsA is gated by pH, opening for conduction under acidic conditions. Molecular determinants responsible for this effect have been identified at the extracellular selectivity filter, at the membrane-cytoplasm interface (TM2 gate), and in the cytoplasmic C-terminal domain (CTD), an amphiphilic four-helix bundle mediated by hydrophobic and electrostatic interactions. Here we have employed NMR and EPR to provide a structural view of the pH-induced open-to-closed CTD transition. KcsA was embedded in lipoprotein nanodiscs (LPNs), selectively methyl-protonated at Leu/Val residues to allow observation of both states by NMR, and spin-labeled for the purposes of EPR studies. We observed a pHinduced structural change between an associated structured CTD at neutral pH and a dissociated flexible CTD at acidic pH, with a transition in the 5.0-5.5 range, consistent with a stabilization of the CTD by channel architecture. A double mutant constitutively open at the TM2 gate exhibited reduced stability of associated CTD, as indicated by weaker spin-spin interactions, a shift to higher transition pH values, and a tenfold reduction in the population of the associated "closed" channels. We extended these findings for isolated CTD-derived peptides to full-length KcsA and have established a contribution of the CTD to KcsA pH-controlled gating, which exhibits a strong correlation with the state of the proximal TM2 gate.

Designer and natural peptide toxin blockers of the KcsA potassium channel identified by phage display.

Peptide neurotoxins are powerful tools for research, diagnosis, and treatment of disease. Limiting broader use, most receptors lack an identified toxin that binds with high affinity and specificity. This paper describes isolation of toxins for one such orphan target, KcsA, a potassium channel that has been fundamental to delineating the structural basis for ion channel function. A phage-display strategy is presented whereby ∼1.5 million novel and natural peptides are fabricated on the scaffold present in ShK, a sea anemone type I (SAK1) toxin stabilized by three disulfide bonds. We describe two toxins selected by sorting on purified KcsA, one novel (Hui1, 34 residues) and one natural (HmK, 35 residues). Hui1 is potent, blocking single KcsA channels in planar lipid bilayers half-maximally (Ki) at 1 nM. Hui1 is also specific, inhibiting KcsA-Shaker channels in Xenopus oocytes with a Ki of 0.5 nM whereas Shaker, Kv1.2, and Kv1.3 channels are blocked over 200-fold less well. HmK is potent but promiscuous, blocking KcsA-Shaker, Shaker, Kv1.2, and Kv1.3 channels with Ki of 1-4 nM. As anticipated, one Hui1 blocks the KcsA pore and two conserved toxin residues, Lys21 and Tyr22, are essential for high-affinity binding. Unexpectedly, potassium ions traversing the channel from the inside confer voltage sensitivity to the Hui1 off-rate via Arg23, indicating that Lys21 is not in the pore. The 3D structure of Hui1 reveals a SAK1 fold, rationalizes KcsA inhibition, and validates the scaffold-based approach for isolation of high-affinity toxins for orphan receptors.

A J-modulated protonless NMR experiment characterizes the conformational ensemble of the intrinsically disordered protein WIP.

Intrinsically disordered proteins (IDPs) are multi-conformational polypeptides that lack a single stable three-dimensional structure. It has become increasingly clear that the versatile IDPs play key roles in a multitude of biological processes, and, given their flexible nature, NMR is a leading method to investigate IDP behavior on the molecular level. Here we present an IDP-tailored J-modulated experiment designed to monitor changes in the conformational ensemble characteristic of IDPs by accurately measuring backbone one- and two-bond J(15N,13Cα) couplings. This concept was realized using a unidirectional (H)NCO 13C-detected experiment suitable for poor spectral dispersion and optimized for maximum coverage of amino acid types. To demonstrate the utility of this approach we applied it to the disordered actin-binding N-terminal domain of WASp interacting protein (WIP), a ubiquitous key modulator of cytoskeletal changes in a range of biological systems. One- and two-bond J(15N,13Cα) couplings were acquired for WIP residues 2-65 at various temperatures, and in denaturing and crowding environments. Under native conditions fitted J-couplings identified in the WIP conformational ensemble a propensity for extended conformation at residues 16-23 and 45-60, and a helical tendency at residues 28-42. These findings are consistent with a previous study of the based upon chemical shift and RDC data and confirm that the WIP2-65 conformational ensemble is biased towards the structure assumed by this fragment in its actin-bound form. The effects of environmental changes upon this ensemble were readily apparent in the J-coupling data, which reflected a significant decrease in structural propensity at higher temperatures, in the presence of 8 M urea, and under the influence of a bacterial cell lysate. The latter suggests that crowding can cause protein unfolding through protein-protein interactions that stabilize the unfolded state. We conclude that J-couplings are a useful measureable in characterizing structural ensembles in IDPs, and that the proposed experiment provides a practical method for accurately performing such measurements, once again emphasizing the power of NMR in studying IDP behavior.

Self-Assembled Cyclic d,l-α-Peptides as Generic Conformational Inhibitors of the α-Synuclein Aggregation and Toxicity: In Vitro and Mechanistic Studies.

Many peptides and proteins with large sequences and structural differences self-assemble into disease-causing amyloids that share very similar biochemical and biophysical characteristics, which may contribute to their cross-interaction. Here, we demonstrate how the self-assembled, cyclic d,l-α-peptide CP-2, which has similar structural and functional properties to those of amyloids, acts as a generic inhibitor of the Parkinson's disease associated α-synuclein (α-syn) aggregation to toxic oligomers by an "off-pathway" mechanism. We show that CP-2 interacts with the N-terminal and the non-amyloid-ß component region of α-syn, which are responsible for α-syn's membrane intercalation and self-assembly, thus changing the overall conformation of α-syn. CP-2 also remodels α-syn fibrils to nontoxic amorphous species and permeates cells through endosomes/lysosomes to reduce the accumulation and toxicity of intracellular α-syn in neuronal cells overexpressing α-syn. Our studies suggest that targeting the common structural conformation of amyloids may be a promising approach for developing new therapeutics for amyloidogenic diseases.

Architecture of the hepatitis C virus E1 glycoprotein transmembrane domain studied by NMR.

Oligomerization of hepatitis C viral envelope proteins E1 and E2 is essential to virus fusion and assembly. Although interactions within the transmembrane (TM) domains of these glycoproteins have proven contributions to the E1/E2 heterodimerization process and consequent infectivity, there is little structural information on this entry mechanism. Here, as a first step towards our long-term goal of understanding the interaction between E1 and E2 TM-domains, we have expressed, purified and characterized E1-TM using structural biomolecular NMR methods. An MBP-fusion expression system yielded sufficient quantities of pure E1-TM, which was solubilized in two membrane-mimicking environments, SDS- and LPPG-micelles, affording samples amenable to NMR studies. Triple resonance assignment experiments and relaxation measurements provided information on the secondary structure and global fold of E1-TM in these environments. In SDS micelles E1-TM adopts a helical conformation, with helical stretches at residues 354-363 and 371-379 separated by a more flexible segment of residues 364-370. In LPPG micelles a helical conformation was observed for residues 354-377 with greater flexibility in the 366-367 dyad, suggesting LPPG provides a more native environment for the peptide. Replacement of key positively charged residue K370 with an alanine did not affect the secondary structure of E1-TM but did change the relative positioning within the micelle of the two helices. These results lay the foundation for structure determination of E1-TM and a molecular understanding of how E1-TM flexibility enhances its interaction with E2-TM during heterodimerization and membrane fusion.

Global fold and backbone dynamics of the hepatitis C virus E2 glycoprotein transmembrane domain determined by NMR.

E1 and E2 are two hepatitis C viral envelope glycoproteins that assemble into a heterodimer that is essential for membrane fusion and penetration into the target cell. Both extracellular and transmembrane (TM) glycoprotein domains contribute to this interaction, but study of TM-TM interactions has been limited because synthesis and structural characterization of these highly hydrophobic segments present significant challenges. In this NMR study, by successful expression and purification of the E2 transmembrane domain as a fusion construct we have determined the global fold and characterized backbone motions for this peptide incorporated in phospholipid micelles. Backbone resonance frequencies, relaxation rates and solvent exposure measurements concur in showing this domain to adopt a helical conformation, with two helical segments spanning residues 717-726 and 732-746 connected by an unstructured linker containing the charged residues D728 and R730 involved in E1 binding. Although this linker exhibits increased local motions on the ps timescale, the dominating contribution to its relaxation is the global tumbling motion with an estimated correlation time of 12.3ns. The positioning of the helix-linker-helix architecture within the mixed micelle was established by paramagnetic NMR spectroscopy and phospholipid-peptide cross relaxation measurements. These indicate that while the helices traverse the hydrophobic interior of the micelle, the linker lies closer to the micelle perimeter to accommodate its charged residues. These results lay the groundwork for structure determination of the E1/E2 complex and a molecular understanding of glycoprotein heterodimerization.

NMR studies of DNA microcapsules prepared using sonochemical methods.

DNA molecules were recently converted using ultrasonic irradiation into microcapsules that can trap hydrophobic molecules in aqueous solution. These DNA microcapsules are capable of penetrating prokaryotic and eukaryotic cells, delivering drugs and transferring genetic information e.g. for protein expression into the host cells. DNA molecules of different sizes and structures can be assembled into spherical capsules, but to date, the interactions that hold them together in these large structural constructs are unknown. In the current study, capsules prepared from a 12 base double helix DNA were investigated using NMR spectroscopy. Solution NMR studies of the DNA emulsion reveal DNA molecules with a perturbed structure with a size similar to the precursor DNA based on diffusion NMR measurements. 2D NMR correlation measurements and chemical shift perturbation analysis show partial unzipping of AT base pairs in the centre of the modified duplex, freeing nucleoside bases to interact with other bases on other precursor molecules thereby facilitating aggregation. Slow tumbling of the microspheres renders them invisible in solution NMR spectra; therefore magic angle spinning NMR measurements are performed which provide limited evidence of the DNA in the microcapsule state.

Conformational flexibility in the binding surface of the potassium channel blocker ShK.

ShK is a 35-residue peptide that binds with high affinity to human voltage-gated potassium channels through a conserved K-Y dyad. Here we have employed NMR measurements of backbone-amide (15)N spin-relaxation rates to investigate motions of the ShK backbone. Although ShK is rigid on the ps to ns timescale, increased linewidths observed for 11 backbone-amide (15)N resonances identify chemical or conformational exchange contributions to the spin relaxation. Relaxation dispersion profiles indicate that exchange between major and minor conformers occurs on the sub-millisecond timescale. Affected residues are mostly clustered around the central helix-kink-helix structure and the critical K22-Y23 motif. We suggest that the less structured minor conformer increases the exposure of Y23, known to contribute to binding affinity and selectivity, thereby facilitating its interaction with potassium channels. These findings have potential implications for the design of new channel blockers based on ShK.

NMR determines transient structure and dynamics in the disordered C-terminal domain of WASp interacting protein.

WASp-interacting protein (WIP) is a 503-residue proline-rich polypeptide expressed in human T cells. The WIP C-terminal domain binds to Wiskott-Aldrich syndrome protein (WASp) and regulates its activation and degradation, and the WIP-WASp interaction has been shown to be critical for actin polymerization and implicated in the onset of WAS and X-linked thrombocytopenia. WIP is predicted to be an intrinsically disordered protein, a class of polypeptides that are of great interest because they violate the traditional structure-function paradigm. In this first (to our knowledge) study of WIP in its unbound state, we used NMR to investigate the biophysical behavior of WIP(C), a C-terminal domain fragment of WIP that includes residues 407-503 and contains the WASp-binding site. In light of the poor spectral dispersion exhibited by WIP(C) and the high occurrence (25%) of proline residues, we employed 5D-NMR(13)C-detected NMR experiments with nonuniform sampling to accomplish full resonance assignment. Secondary chemical-shift analysis, (15)N relaxation rates, and protection from solvent exchange all concurred in detecting transient structure located in motifs that span the WASp-binding site. Residues 446-456 exhibited a propensity for helical conformation, and an extended conformation followed by a short, capped helix was observed for residues 468-478. The (13)C-detected approach allows chemical-shift assignment in the WIP(C) polyproline stretches and thus sheds light on their conformation and dynamics. The effects of temperature on chemical shifts referenced to a denatured sample of the polypeptide demonstrate that heating reduces the structural character of WIP(C). Thus, we conclude that the disordered WIP(C) fragment is comprised of regions with latent structure connected by flexible loops, an architecture with implications for binding affinity and function.

A novel Pyk2-derived peptide inhibits invadopodia-mediated breast cancer metastasis.

Dissemination of cancer cells from the primary tumor into distant body tissues and organs is the leading cause of death in cancer patients. While most clinical strategies aim to reduce or impede the growth of the primary tumor, no treatment to eradicate metastatic cancer exists at present. Metastasis is mediated by feet-like cytoskeletal structures called invadopodia which allow cells to penetrate through the basement membrane and intravasate into blood vessels during their spread to distant tissues and organs. The non-receptor tyrosine kinase Pyk2 is highly expressed in breast cancer, where it mediates invadopodia formation and function via interaction with the actin-nucleation-promoting factor cortactin. Here, we designed a cell-permeable peptide inhibitor that contains the second proline-rich region (PRR2) sequence of Pyk2, which binds to the SH3 domain of cortactin and inhibits the interaction between Pyk2 and cortactin in invadopodia. The Pyk2-PRR2 peptide blocks spontaneous lung metastasis in immune-competent mice by inhibiting cortactin tyrosine phosphorylation and actin polymerization-mediated maturation and activation of invadopodia, leading to reduced MMP-dependent tumor cell invasiveness. The native structure of the Pyk2-PRR2:cortactin-SH3 complex was determined using nuclear magnetic resonance (NMR), revealing an extended class II interaction surface spanning the canonical binding groove and a second hydrophobic surface which significantly contributes to ligand affinity. Using structure-guided design, we created a mutant peptide lacking critical residues involved in binding that failed to inhibit invadopodia maturation and function and consequent metastatic dissemination in mice. Our findings shed light on the specific molecular interactions between Pyk2 and cortactin and may lead to the development of novel strategies for preventing dissemination of primary breast tumors predicted at the time of diagnosis to be highly metastatic, and of secondary tumors that have already spread to other parts of the body.

Biomolecular condensates formed by designer minimalistic peptides.

Inspired by the role of intracellular liquid-liquid phase separation (LLPS) in formation of membraneless organelles, there is great interest in developing dynamic compartments formed by LLPS of intrinsically disordered proteins (IDPs) or short peptides. However, the molecular mechanisms underlying the formation of biomolecular condensates have not been fully elucidated, rendering on-demand design of synthetic condensates with tailored physico-chemical functionalities a significant challenge. To address this need, here we design a library of LLPS-promoting peptide building blocks composed of various assembly domains. We show that the LLPS propensity, dynamics, and encapsulation efficiency of compartments can be tuned by changes to the peptide composition. Specifically, with the aid of Raman and NMR spectroscopy, we show that interactions between arginine and aromatic amino acids underlie droplet formation, and that both intra- and intermolecular interactions dictate droplet dynamics. The resulting sequence-structure-function correlation could support the future development of compartments for a variety of applications.

4D non-uniformly sampled HCBCACON and ¹J(NCα)-selective HCBCANCO experiments for the sequential assignment and chemical shift analysis of intrinsically disordered proteins.

A pair of 4D NMR experiments for the backbone assignment of disordered proteins is presented. The experiments exploit (13)C direct detection and non-uniform sampling of the indirectly detected dimensions, and provide correlations of the aliphatic proton (H(α), and H(ß)) and carbon (C(α), C(ß)) resonance frequencies to the protein backbone. Thus, all the chemical shifts regularly used to map the transient secondary structure motifs in the intrinsically disordered proteins (H(α), C(α), C(ß), C', and N) can be extracted from each spectrum. Compared to the commonly used assignment strategy based on matching the C(α) and C(ß) chemical shifts, inclusion of the H(α) and H(ß) provides up to three extra resonance frequencies that decrease the chance of ambiguous assignment. The experiments were successfully applied to the original assignment of a 12.8 kDa intrinsically disordered protein having a high content of proline residues (26 %) in the sequence.

The Disordered Cellular Multi-Tasker WIP and Its Protein-Protein Interactions: A Structural View.

WASp-interacting protein (WIP), a regulator of actin cytoskeleton assembly and remodeling, is a cellular multi-tasker and a key member of a network of protein-protein interactions, with significant impact on health and disease. Here, we attempt to complement the well-established understanding of WIP function from cell biology studies, summarized in several reviews, with a structural description of WIP interactions, highlighting works that present a molecular view of WIP's protein-protein interactions. This provides a deeper understanding of the mechanisms by which WIP mediates its biological functions. The fully disordered WIP also serves as an intriguing example of how intrinsically disordered proteins (IDPs) exert their function. WIP consists of consecutive small functional domains and motifs that interact with a host of cellular partners, with a striking preponderance of proline-rich motif capable of interactions with several well-recognized binding partners; indeed, over 30% of the WIP primary structure are proline residues. We focus on the binding motifs and binding interfaces of three important WIP segments, the actin-binding N-terminal domain, the central domain that binds SH3 domains of various interaction partners, and the WASp-binding C-terminal domain. Beyond the obvious importance of a more fundamental understanding of the biology of this central cellular player, this approach carries an immediate and highly beneficial effect on drug-design efforts targeting WIP and its binding partners. These factors make the value of such structural studies, challenging as they are, readily apparent.

Tethered peptide neurotoxins display two blocking mechanisms in the K+ channel pore as do their untethered analogs.

We show here that membrane-tethered toxins facilitate the biophysical study of the roles of toxin residues in K+ channel blockade to reveal two blocking mechanisms in the K+ channel pore. The structure of the sea anemone type I (SAK1) toxin HmK is determined by NMR. T-HmK residues are scanned by point mutation to map the toxin surface, and seven residues are identified to be critical to occlusion of the KcsA channel pore. T-HmK-Lys22 is shown to interact with K+ ions traversing the KcsA pore from the cytoplasm conferring voltage dependence on the toxin off rate, a classic mechanism that we observe as well with HmK in solution and for Kv1.3 channels. In contrast, two related SAK1 toxins, Hui1 and ShK, block KcsA and Kv1.3, respectively, via an arginine rather than the canonical lysine, when tethered and as free peptides.

Synthesis and Characterization of Poly(RGD) Proteinoid Polymers and NIR Fluorescent Nanoparticles of Optimal d,l-Configuration for Drug-Delivery Applications-In Vitro Study.

RGD sequence is a tripeptide composed of three amino acids: arginine (R), glycine (G), and aspartic acid (D). The RGD peptide has a high affinity to the integrin alpha v beta 3, which is overexpressed on the membrane of many cancer cells and is attracted to areas of angiogenesis. Proteinoids are biodegradable polymers based on amino acids which are formed by bulk thermal step-growth polymerization mechanism. Hollow proteinoid nanoparticles (NPs) may be formed via self-assembly process of the proteinoid polymers. We propose using novel RGD-based proteinoid polymers to manufacture NPs in which the RGD motif is self-incorporated in the proteinoid backbone. Such P(RGD) NPs can act both as a drug carrier (by encapsulation of a desired drug) and as a targeting delivery system. This article presents the synthesis of four RGD proteinoids with different RGD optical configurations, (d) or (l) arginine, glycine, and (d) or (l) aspartic acid, in order to determine which configuration is optimal as a drug-targeting carrier. These new RGD proteinoid polymers possess high molecular weights and molecular weight monodispersity. Homonuclear nuclear magnetic resonance methods were employed to predict the expected concentration of RGD tripeptide sequence in the polymer. Near infrared fluorescent NPs have been prepared by the encapsulation of indocyanine green (ICG) dye within the different P(RGD) NPs. The dry diameters of the hollow P(RdGDd), P(RdGD), P(RGD), and P(RGDd) NPs are 55 ± 13, 48 ± 9, 45 ± 11, and 42 ± 9 nm, respectively, whereas those of the ICG-encapsulated NPs were significantly higher, 141 ± 24, 95 ± 13, 86 ± 11, and 87 ± 12 nm, respectively. The ICG-encapsulated P(RdGD) NPs exhibited higher selectivity toward epithelial injury, as demonstrated using an in vitro scratch assay, because the P(RdGD) NPs accumulated in the injured area at higher concentrations when compared to other P(RGD) NPs with different chiralities. Therefore, the P(RdGD) polymer configuration is the polymer of choice for use as a targeted drug carrier to areas of angiogenesis, such as in tumors, wounds, or cuts.

Engineering of Durable Antifog Thin Coatings on Plastic Films by UV-Curing of Proteinoid Prepolymers with PEG-Diacrylate Monomers.

Fog formation on transparent surfaces constitutes a major challenge in several optical applications, such as plastic packaging, lenses, mirrors, and windshields. To overcome this problem, we prepared and characterized durable antifog thin coatings on plastic films such as polyethylene terephthalate (PET). Proteinoids are biocompatible random polymers made of α-amino acids by thermal step-growth polymerization. Proteinoid prepolymers were prepared by adding activated double bonds to proteinoids via the Michael addition reaction. A series of thin antifog cross-linked coatings were prepared by spreading on PET films with a Mayer rod various mixtures of the proteinoid prepolymers, polyethylene glycol diacrylate, and a photoinitiator, followed by UV-curing of the dried coatings. The antifog properties of the coatings were determined by the contact angle, roughness, haze, and gloss measurements, as well as hot and cold fog tests, to examine the optical properties of the films under fog formation conditions. Mechanical properties such as adhesion, robustness, and abrasion resistance of the antifog coatings were examined by tape, knife-scratch, and sandpaper abrasion tests. The effect of coating composition, wettability, and roughness on the antifog properties of the coated PET films was elucidated. The formula was optimized, and the corresponding UV-cured antifog cross-linked thin coating exhibited transparency with good adhesion and excellent durable antifog performance.

Folding of unstructured peptoids and formation of hetero-bimetallic peptoid complexes upon side-chain-to-metal coordination.

Helices are key structural features in biopolymers, enabling a variety of biological functions. Mimicking these secondary structure motifs has wide potential in the development of biomimetic materials. Peptoids, N-substituted glycine oligomers, are an important class of peptide mimics that can adopt polyproline type helices if the majority of their sequence consists of chiral bulky pendent groups. Such side-chains are structure inducers but they have no functional value. We present here the inclusion of several metal-binding groups in one peptoid oligomer as a new platform towards the development of functional helical peptoids. Thus, we describe the coordination of two metal ions to unstructured peptoids incorporating four 8-hydroxyquinoline (HQ) ligands at fixed positions as two (HQ, HQ) metal binding sites, and a mixture of chiral benzyl and alkyl substituents in varied positions along the peptoid backbone. For the first time, we demonstrate by circular dichroism spectroscopy, solution NMR techniques and high-level DFT calculations that some of these unstructured peptoids can fold upon metal binding to form helical structures. Replacing one HQ ligand with a terpyridine (Terpy) ligand resulted in unique sequences that can selectively coordinate Cu2+ to the (Terpy, HQ) and Zn2+ (or Co2+) to the (HQ, HQ) sites from a solution mixture containing Cu2+ and Zn2+ (or Co2+) ions. Interestingly, the binding of Cu2+ to the (Terpy, HQ) site in one of these peptoids can initiate a conformational change that in turn facilitates the coordination of Zn2+ (or Co2+) ions to the (HQ, HQ) site, demonstrating a unique example of positive allosteric cooperativity in peptide mimics.