Differences in heavy metal binding to cysteine-containing coiled-coil peptides.

One third of all structurally characterised proteins contain a metal; however, the interplay between metal-binding and peptide/protein folding has yet to be fully elucidated. To better understand how metal binding affects peptide folding, a range of metals should be studied within a specific scaffold. To this end, we modified a histidine-containing coiled-coil peptide to create a cysteine-containing scaffold, named CX3C, which was designed to bind heavy metal ions. In addition, we generated a peptide named CX2C, which contains a binding site more commonly found in natural proteins. Using a combination of analytical techniques including circular dichroism (CD) spectroscopy, UV-Vis spectroscopy and size-exclusion chromatography coupled to multi-angle light scattering (SEC-MALS), we examined the differences in the metal-binding properties of the two peptides. Both peptides are largely unfolded in the apo state due to the disruption of the hydrophobic core by inclusion of the polar cysteine residues. However, this unfolding is overcome by the addition of Cd(II), Pb(II) and Hg(II), and helical assemblies are formed. Both peptides have differing affinities for these metal ions, a fact likely attributed to the differing sizes of the ions. We also show that the oligomerisation state of the peptide complexes and the coordination geometries of the metal ions differ between the two peptide scaffolds. These findings highlight that subtle changes in the primary structure of a peptide can have considerable implications for metal binding.

Efficient mRNA delivery using lipid nanoparticles modified with fusogenic coiled-coil peptides.

Gene delivery has great potential in modulating protein expression in specific cells to treat diseases. Such therapeutic gene delivery demands sufficient cellular internalization and endosomal escape. Of various nonviral nucleic acid delivery systems, lipid nanoparticles (LNPs) are the most advanced, but still, are very inefficient as the majority are unable to escape from endosomes/lysosomes. Here, we develop a highly efficient gene delivery system using fusogenic coiled-coil peptides. We modified LNPs, carrying EGFP-mRNA, and cells with complementary coiled-coil lipopeptides. Coiled-coil formation between these lipopeptides induced fast nucleic acid uptake and enhanced GFP expression. The cellular uptake of coiled-coil modified LNPs is likely driven by membrane fusion thereby omitting typical endocytosis pathways. This direct cytosolic delivery circumvents the problems commonly observed with the limited endosomal escape of mRNA. Therefore fusogenic coiled-coil peptide modification of existing LNP formulations to enhance nucleic acid delivery efficiency could be beneficial for several gene therapy applications.

Enhanced activity against a third-generation cephalosporin by destabilization of the active site of a class A beta-lactamase.

The ß-lactamase BlaC conveys resistance to a broad spectrum of ß-lactam antibiotics to its host Mycobacterium tuberculosis but poorly hydrolyzes third-generation cephalosporins, such as ceftazidime. Variants of other ß-lactamases have been reported to gain activity against ceftazidime at the cost of the native activity. To understand this trade-off, laboratory evolution was performed, screening for enhanced ceftazidime activity. The variant BlaC Pro167Ser shows faster breakdown of ceftazidime, poor hydrolysis of ampicillin and only moderately reduced activity against nitrocefin. NMR spectroscopy, crystallography and kinetic assays demonstrate that the resting state of BlaC P167S exists in an open and a closed state. The open state is more active in the hydrolysis of ceftazidime. In this state the catalytic residue Glu166, generally believed to be involved in the activation of the water molecule required for deacylation, is rotated away from the active site, suggesting it plays no role in the hydrolysis of ceftazidime. In the closed state, deacylation of the BlaC-ceftazidime adduct is slow, while hydrolysis of nitrocefin, which requires the presence of Glu166 in the active site, is barely affected, providing a structural explanation for the trade-off in activities.

Enhanced Liposomal Drug Delivery Via Membrane Fusion Triggered by Dimeric Coiled-Coil Peptides.

An ideal nanomedicine system improves the therapeutic efficacy of drugs. However, most nanomedicines enter cells via endosomal/lysosomal pathways and only a small fraction of the cargo enters the cytosol inducing therapeutic effects. To circumvent this inefficiency, alternative approaches are desired. Inspired by fusion machinery found in nature, synthetic lipidated peptide pair E4/K4 is used to induce membrane fusion previously. Peptide K4 interacts specifically with E4, and it has a lipid membrane affinity and resulting in membrane remodeling. To design efficient fusogens with multiple interactions, dimeric K4 variants are synthesized to improve fusion with E4-modified liposomes and cells. The secondary structure and self-assembly of dimers are studied; the parallel PK4 dimer forms temperature-dependent higher-order assemblies, while linear K4 dimers form tetramer-like homodimers. The structures and membrane interactions of PK4 are supported by molecular dynamics simulations. Upon addition of E4, PK4 induced the strongest coiled-coil interaction resulting in a higher liposomal delivery compared to linear dimers and monomer. Using a wide spectrum of endocytosis inhibitors, membrane fusion is found to be the main cellular uptake pathway. Doxorubicin delivery results in efficient cellular uptake and concomitant antitumor efficacy. These findings aid the development of efficient delivery systems of drugs into cells using liposome-cell fusion strategies.

SNARE mimic peptide triggered membrane fusion kinetics revealed using single particle techniques.

Membrane fusion is an essential part of the proper functioning of life. As such it is not only important that organisms carefully regulate the process, but also that it is well understood. One way to facilitate and study membrane fusion is to use artificial, minimalist, fusion peptides. In this study the efficiency and kinetics of two fusion peptides, denoted CPE and CPK, were studied using single-particle TIRF microscopy. CPE and CPK are helical peptides which interact with each other, forming a coiled-coil motif. The peptides can be inserted into a lipid membrane using a lipid anchor, and if these peptides are anchored in opposing lipid membranes, then the coiled-coil interaction can provide the mechanical force necessary to overcome the energy barrier to initiate fusion, much in the same way the SNARE complex does. In this study we find that the fusogenic facilitation of CPE and CPK in liposomes is, at least partially, dependent on the size of the particle. In addition, under certain fusogenic conditions such as when using small liposomes of ∼60 nm in diameter, CPK alone is enough to facilitate membrane fusion in both bulk and single-particle studies. We show this using bulk lipid mixing assays utilizing FRET and single-particle TIRF, making use of dequenching fluorophores to indicate fusion. This provides us with new insights into the mechanisms of peptide-mediated membrane fusion and illuminates both challenges as well as opportunities when designing drug delivery systems.

Azobenzene-Based Amino Acids for the Photocontrol of Coiled-Coil Peptides.

Coiled-coil peptides are high-affinity, selective, self-assembling binding motifs, making them attractive components for the preparation of functional biomaterials. Photocontrol of coiled-coil self-assembly allows for the precise localization of their activity. To rationally explore photoactivity in a model coiled coil, three azobenzene-containing amino acids were prepared and substituted into the hydrophobic core of the E3/K3 coiled-coil heterodimer. Two of the non-natural amino acids, APhe1 and APhe2, are based on phenylalanine and differ in the presence of a carboxylic acid group. These have previously been demonstrated to modulate protein activity. When incorporated into peptide K3, coiled-coil binding strength was affected upon isomerization, with the two variants differing in their most folded state. The third azobenzene-containing amino acid, APgly, is based on phenylglycine and was prepared to investigate the effect of amino acid size on photoisomerization. When APgly is incorporated into the coiled coil, a 4.7-fold decrease in folding constant is observed upon trans-to-cis isomerization─the largest difference for all three amino acids. Omitting the methylene group between azobenzene and α-carbon was theorized to both position the diazene of APgly closer to the hydrophobic amino acids and reduce the possible rotations of the amino acid, with molecular dynamics simulations supporting these hypotheses. These results demonstrate the ability of photoswitchable amino acids to control coiled-coil assembly through disruption of the hydrophobic interface, a strategy that should be widely applicable.

Gold nanoparticles decorated with ovalbumin-derived epitopes: effect of shape and size on T-cell immune responses.

Gold nanoparticles (GNPs) can be manufactured in various shapes, and their size is programmable, which permits the study of the effects imposed by these parameters on biological processes. However, there is currently no clear evidence that a certain shape or size is beneficial. To address this issue, we have utilised GNPs and gold nanorods (GNRs) functionalised with model epitopes derived from chicken ovalbumin (OVA257-264 and OVA323-339). By using two distinct epitopes, it was possible to draw conclusions regarding the impact of nanoparticle shape and size on different aspects of the immune response. Our findings indicate that the peptide amphiphile-coated GNPs and GNRs are a safe and versatile epitope-presenting system. Smaller GNPs (∼15 nm in diameter) induce significantly less intense T-cell responses. Furthermore, effective antigen presentation via MHC-I was observed for larger spherical particles (∼40 nm in diameter), and to a lesser extent for rod-like particles (40 by 15 nm). At the same time, antigen presentation via MHC-II strongly correlated with the cellular uptake, with smaller GNPs being the least efficient. We believe these findings will have implications for vaccine development, and lead to a better understanding of cellular uptake and antigen egress from lysosomes into the cytosol.

Coating gold nanorods with self-assembling peptide amphiphiles promotes stability and facilitates in vivo two-photon imaging.

Gold nanorods (GNRs) are versatile asymmetric nanoparticles with unique optical properties. These properties make GNRs ideal agents for applications such as photothermal cancer therapy, biosensing, and in vivo imaging. However, as-synthesised GNRs need to be modified with a biocompatible stabilising coating in order to be employed in these fields as the ligands used to stabilise GNRs during synthesis are toxic. An issue is that GNR performance in the aforementioned techniques can be affected by these modified coatings. For example if coatings are too thick then GNR entry into cells, or their sensitivity in sensing applications, can be compromised. Here we show that thiolated peptide amphiphiles (PAs) can act as GNR stabilisers and provide a thin and highly-stable coating under physiologically relevant conditions. Additionally, all tested PAs formed highly ordered (51.8-58.8% ß-content), and dense (2.62-3.87 peptides per nm2) monolayers on the GNR surface. Moreover, the PA-coated GNRs demonstrated no cytotoxicity in vitro and, via injection in zebrafish embryos, the behavior and cellular interactions of such PA-coated GNRs were visualised in vivo, in real time, with two-photon (2P) microscopy.

Controlling amphipathic peptide adsorption by smart switchable germanium interfaces.

The in situ control of reversible protein adsorption to a surface is a critical step towards biofouling prevention and finds utilisation in bioanalytical applications. In this work, adsorption of peptides is controlled by employing the electrode potential induced, reversible change of germanium (100) surface termination between a hydrophobic, hydrogen terminated and a hydrophilic, hydroxyl terminated surface. This simple but effective 'smart' interface is used to direct adsorption of two peptides models, representing the naturally highly abundant structural motifs of amphipathic helices and coiled-coils. Their structural similarity coincides with their opposite overall charge and hence allows the examination of the influence of charge and hydrophobicity on adsorption. Polarized attenuated total reflection infrared (ATR-IR) spectroscopy at controlled electrode potential has been used to follow the adsorption process at physiological pH in deuterated buffer. The delicate balance of hydrophobic and electrostatic peptide/surface interactions leads to two different processes upon switching that are both observed in situ: reversible adsorption and reversible reorientation. Negatively charged peptide adsorption can be fully controlled by switching to the hydrophobic interface, while the same switch causes the positively charged, helical peptide to tilt down. This principle can be used for 'smart' adsorption control of a wider variety of proteins and peptides and hence find application, as e.g. a bioanalytical tool or functional biosensor.

Liposome fusion with orthogonal coiled coil peptides as fusogens: the efficacy of roleplaying peptides.

Biological membrane fusion is a highly specific and coordinated process as a multitude of vesicular fusion events proceed simultaneously in a complex environment with minimal off-target delivery. In this study, we develop a liposomal fusion model system with specific recognition using lipidated derivatives of a set of four de novo designed heterodimeric coiled coil (CC) peptide pairs. Content mixing was only obtained between liposomes functionalized with complementary peptides, demonstrating both fusogenic activity of CC peptides and the specificity of this model system. The diverse peptide fusogens revealed important relationships between the fusogenic efficacy and the peptide characteristics. The fusion efficiency increased from 20% to 70% as affinity between complementary peptides decreased, (from K F ≈ 108 to 104 M-1), and fusion efficiency also increased due to more pronounced asymmetric role-playing of membrane interacting 'K' peptides and homodimer-forming 'E' peptides. Furthermore, a new and highly fusogenic CC pair (E3/P1K) was discovered, providing an orthogonal peptide triad with the fusogenic CC pairs P2E/P2K and P3E/P3K. This E3/P1k pair was revealed, via molecular dynamics simulations, to have a shifted heptad repeat that can accommodate mismatched asparagine residues. These results will have broad implications not only for the fundamental understanding of CC design and how asparagine residues can be accommodated within the hydrophobic core, but also for drug delivery systems by revealing the necessary interplay of efficient peptide fusogens and enabling the targeted delivery of different carrier vesicles at various peptide-functionalized locations.

Novel anti-repression mechanism of H-NS proteins by a phage protein.

H-NS family proteins, bacterial xenogeneic silencers, play central roles in genome organization and in the regulation of foreign genes. It is thought that gene repression is directly dependent on the DNA binding modes of H-NS family proteins. These proteins form lateral protofilaments along DNA. Under specific environmental conditions they switch to bridging two DNA duplexes. This switching is a direct effect of environmental conditions on electrostatic interactions between the oppositely charged DNA binding and N-terminal domains of H-NS proteins. The Pseudomonas lytic phage LUZ24 encodes the protein gp4, which modulates the DNA binding and function of the H-NS family protein MvaT of Pseudomonas aeruginosa. However, the mechanism by which gp4 affects MvaT activity remains elusive. In this study, we show that gp4 specifically interferes with the formation and stability of the bridged MvaT-DNA complex. Structural investigations suggest that gp4 acts as an 'electrostatic zipper' between the oppositely charged domains of MvaT protomers, and stabilizes a structure resembling their 'half-open' conformation, resulting in relief of gene silencing and adverse effects on P. aeruginosa growth. The ability to control H-NS conformation and thereby its impact on global gene regulation and growth might open new avenues to fight Pseudomonas multidrug resistance.

Magnetic-Activated Cell Sorting Using Coiled-Coil Peptides: An Alternative Strategy for Isolating Cells with High Efficiency and Specificity.

Magnetic-activated cell sorting (MACS) is an affinity-based technique used to separate cells according to the presence of specific markers. Current MACS systems generally require an antigen to be expressed at the cell surface; these antigen-presenting cells subsequently interact with antibody-labeled magnetic particles, facilitating separation. Here, we present an alternative MACS method based on coiled-coil peptide interactions. We demonstrate that HeLa, CHO, and NIH3T3 cells can either incorporate a lipid-modified coiled-coil-forming peptide into their membrane, or that the cells can be transfected with a plasmid containing a gene encoding a coiled-coil-forming peptide. Iron oxide particles are functionalized with the complementary peptide and, upon incubation with the cells, labeled cells are facilely separated from nonlabeled populations. In addition, the resulting cells and particles can be treated with trypsin to facilitate detachment of the cells from the particles. Therefore, our new MACS method promotes efficient cell sorting of different cell lines, without the need for antigen presentation, and enables simple detachment of the magnetic particles from cells after the sorting process. Such a system can be applied to rapidly developing, sensitive research areas, such as the separation of genetically modified cells from their unmodified counterparts.

Probing the E/K Peptide Coiled-Coil Assembly by Double Electron-Electron Resonance and Circular Dichroism.

Double electron-electron resonance (DEER, also known as PELDOR) and circular dichroism (CD) spectroscopies were explored for the purpose of studying the specificity of the conformation of peptides induced by their assembly into a self-recognizing system. The E and K peptides are known to form a coiled-coil heterodimer. Two paramagnetic TOAC α-amino acid residues were incorporated into each of the peptides (denoted as K** and E**), and a three-dimensional structural investigation in the presence or absence of their unlabeled counterparts E and K was performed. The TOAC spin-labels, replacing two Ala residues in each compound, are covalently and quasi-rigidly connected to the peptide backbone. They are known not to disturb the native structure, so that any conformational change can easily be monitored and assigned. DEER spectroscopy enables the measurement of the intramolecular electron spin-spin distance distribution between the two TOAC labels, within a length range of 1.5-8 nm. This method allows the individual conformational changes for the K**, K**/E, E**, and E**/K molecules to be investigated in glassy frozen solutions. Our data reveal that the conformations of the E** and K** peptides are strongly influenced by the presence of their counterparts. The results are discussed with those from CD spectroscopy and with reference to the already reported nuclear magnetic resonance data. We conclude that the combined DEER/TOAC approach allows us to obtain accurate and reliable information about the conformation of the peptides before and after their assembly into coiled-coil heterodimers. Applications of this induced fit method to other two-component, but more complex, systems, like a receptor and antagonists, a receptor and a hormone, and an enzyme and a ligand, are discussed.

One Peptide for Them All: Gold Nanoparticles of Different Sizes Are Stabilized by a Common Peptide Amphiphile.

The functionalization of gold nanoparticles (GNPs) with peptidic moieties can prevent their aggregation and facilitate their use for applications both in vitro and in vivo. To date, no peptide-based coating has been shown to stabilize GNPs larger than 30 nm in diameter; such particles are of interest for applications including vaccine development, drug delivery, and sensing. Here, GNPs with diameters of 20, 40, and 100 nm are functionalized with peptide amphiphiles. Using a combination of transmission electron microscopy, UV-vis spectroscopy, and dynamic light scattering, we show that GNPs up to 100 nm in size can be stabilized by these molecules. Moreover, we demonstrate that these peptide amphiphiles form curvature-dependent, ordered structures on the surface of the GNPs and that the GNPs remain disperse at high-salt concentrations and in the presence of competing thiol-containing molecules. These results represent the development of a peptide amphiphile-based coating system for GNPs which has the potential to be beneficial for a wide range of biological applications, in addition to image enhancement and catalysis.

Complement Receptor Targeted Liposomes Encapsulating the Liver X Receptor Agonist GW3965 Accumulate in and Stabilize Atherosclerotic Plaques.

Atherosclerosis is characterized by the retention of lipids in foam cells in the arterial intima. The liver X receptor (LXR) agonist GW3965 is a promising therapeutic compound, since it induces reverse cholesterol transport in foam cells. However, hepatic LXR activation increases plasma and liver lipid levels, inhibiting its clinical development. Herein, a formulation that specifically enhances GW3965 deposition in the atherosclerotic lesion is aimed to be developed. GW3965 is encapsulated in liposomes functionalized with the cyclic peptide Lyp-1 (CGNKRTRGC), which binds the p32 receptor expressed on foam cells. These liposomes show preferential uptake by foam cells in vitro and higher accumulation in atherosclerotic plaques in mice compared to non-targeted liposomes as determined by in vivo imaging. Flow cytometry analysis of plaques reveals increased retention of Lyp-1 liposomes in atherosclerotic plaque macrophages compared to controls (p < 0.05). Long term treatment of established plaques in LDLR -/- mice with GW3965-containing Lyp-1 liposomes significantly reduces plaque macrophage content by 50% (p < 0.01). Importantly, GW3965-containing Lyp-1 liposomes do not increase plasma or hepatic lipid content. Thus, GW3965-containing Lyp-1 liposomes successfully target the atherosclerotic macrophages allowing plaque stabilization without commonly observed side effects of LXR agonists.

Controlled Peptide-Mediated Vesicle Fusion Assessed by Simultaneous Dual-Colour Time-Lapsed Fluorescence Microscopy.

We have employed a model system, inspired by SNARE proteins, to facilitate membrane fusion between Giant Unilamellar Vesicles (GUVs) and Large Unilamellar Vesicles (LUVs) under physiological conditions. In this system, two synthetic lipopeptide constructs comprising the coiled-coil heterodimer-forming peptides K4, (KIAALKE)4, or E4, (EIAALEK)4, a PEG spacer of variable length, and a cholesterol moiety to anchor the peptides into the liposome membrane replace the natural SNARE proteins. GUVs are functionalized with one of the lipopeptide constructs and the fusion process is triggered by adding LUVs bearing the complementary lipopeptide. Dual-colour time lapse fluorescence microscopy was used to visualize lipid- and content-mixing. Using conventional confocal microscopy, lipid mixing was observed on the lipid bilayer of individual GUVs. In addition to lipid-mixing, content-mixing assays showed a low efficiency due to clustering of K4-functionalized LUVs on the GUVs target membranes. We showed that, through the use of the non-ionic surfactant Tween 20, content-mixing between GUVs and LUVs could be improved, meaning this system has the potential to be employed for drug delivery in biological systems.

Modulation of Coiled-Coil Binding Strength and Fusogenicity through Peptide Stapling.

Peptide stapling is a technique which has been widely employed to constrain the conformation of peptides. One of the effects of such a constraint can be to modulate the interaction of the peptide with a binding partner. Here, a cysteine bis-alkylation stapling technique was applied to generate structurally isomeric peptide variants of a heterodimeric coiled-coil forming peptide. These stapled variants differed in the position and size of the formed macrocycle. C-terminal stapling showed the most significant changes in peptide structure and stability, with calorimetric binding analysis showing a significant reduction of binding entropy for stapled variants. This entropy reduction was dependent on cross-linker size and was accompanied by a change in binding enthalpy, illustrating the effects of preorganization. The stapled peptide, along with its binding partner, were subsequently employed as fusogens in a liposome model system. An increase in both lipid- and content-mixing was observed for one of the stapled peptide variants: this increased fusogenicity was attributed to increased coiled-coil binding but not to membrane affinity, an interaction theorized to be a primary driving force in this fusion system.

Selective coordination of three transition metal ions within a coiled-coil peptide scaffold.

Designing peptides that fold and assemble in response to metal ions tests our understanding of how peptide folding and metal binding influence one another. Here, histidine residues are introduced into the hydrophobic core of a coiled-coil trimer, generating a peptide that self-assembles upon the addition of metal ions. HisAD, the resulting peptide, is unstructured in the absence of metal and folds selectively to form an α-helical construct upon complexation with Cu(ii) and Ni(ii) but not Co(ii) or Zn(ii). The structure, and metal-binding ability, of HisAD is probed using a combination of circular dichroism (CD) spectroscopy, analytical ultracentrifugation (AUC), nuclear magnetic resonance (NMR) spectroscopy and X-ray crystallography. These show the peptide is trimeric and binds to both Cu(ii) and Ni(ii) in a 1 : 1 ratio with the histidine residues involved in the metal coordination, as designed. The X-ray crystal structure of the HisAD-Cu(ii) complex reveals the trimeric HisAD peptide coordinates three Cu(ii) ions; this is the first example of such a structure. Additionally, HisAD demonstrates an unprecedented discrimination between transition metal ions, the basis of which is likely to be related to the stability of the peptide-metal complexes formed.

Insights into IgM-mediated complement activation based on in situ structures of IgM-C1-C4b.

Antigen binding by serum Ig-M (IgM) protects against microbial infections and helps to prevent autoimmunity, but causes life-threatening diseases when mistargeted. How antigen-bound IgM activates complement-immune responses remains unclear. We present cryoelectron tomography structures of IgM, C1, and C4b complexes formed on antigen-bearing lipid membranes by normal human serum at 4 °C. The IgM-C1-C4b complexes revealed C4b product release as the temperature-limiting step in complement activation. Both IgM hexamers and pentamers adopted hexagonal, dome-shaped structures with Fab pairs, dimerized by hinge domains, bound to surface antigens that support a platform of Fc regions. C1 binds IgM through widely spread C1q-collagen helices, with C1r proteases pointing outward and C1s bending downward and interacting with surface-attached C4b, which further interacts with the adjacent IgM-Fab2 and globular C1q-recognition unit. Based on these data, we present mechanistic models for antibody-mediated, C1q-transmitted activation of C1 and for C4b deposition, while further conformational rearrangements are required to form C3 convertases.

Designing stable, hierarchical peptide fibers from block co-polypeptide sequences.

Natural materials, such as collagen, can assemble with multiple levels of organization in solution. Achieving a similar degree of control over morphology, stability and hierarchical organization with equilibrium synthetic materials remains elusive. For the assembly of peptidic materials the process is controlled by a complex interplay between hydrophobic interactions, electrostatics and secondary structure formation. Consequently, fine tuning the thermodynamics and kinetics of assembly remains extremely challenging. Here, we synthesized a set of block co polypeptides with varying hydrophobicity and ability to form secondary structure. From this set we select a sequence with balanced interactions that results in the formation of high-aspect ratio thermodynamically favored nanotubes, stable between pH 2 and 12 and up to 80 °C. This stability permits their hierarchical assembly into bundled nanotube fibers by directing the pH and inducing complementary zwitterionic charge behavior. This block co-polypeptide design strategy, using defined sequences, provides a straightforward approach to creating complex hierarchical peptide-based assemblies with tunable interactions.