Hana Robson Marsden

A Reduced SNARE Model for Membrane Fusion

Lets get together: A minimal model system was developed to mimic the SNARE-protein-mediated fusion of biological membranes (see picture). Fusion between two populations of liposomes is controlled by a pair of complementary lipidated oligopeptides that form noncovalent coiled-coil complexes and thereby force the membranes into close proximity to promote fusion. The model system displays the key characteristics of in vivo fusion events.

Self-assembly of coiled coils in synthetic biology: inspiration and progress.

Biological self-assembly is very complex and results in highly functional materials. In effect, it takes a bottom-up approach using biomolecular building blocks of precisely defined shape, size, hydrophobicity, and spatial distribution of functionality. Inspired by, and drawing lessons from self-assembly processes in nature, scientists are learning how to control the balance of many small forces to increase the complexity and functionality of self-assembled nanomaterials. The coiled-coil motif, a multipurpose building block commonly found in nature, has great potential in synthetic biology. In this review we examine the roles that the coiled-coil peptide motif plays in self-assembly in nature, and then summarize the advances that this has inspired in the creation of functional units, assemblies, and systems.

Noncovalent triblock copolymers based on a coiled-coil peptide motif.

The formation of a noncovalent triblock copolymer based on a coiled-coil peptide motif is demonstrated in solution. A specific peptide pair (E and K) able to assemble into heterocoiled coils was chosen as the middle block of the polymer and conjugated to poly(ethylene glycol) (PEG) and polystyrene (PS) as the outer blocks. Mixing equimolar amounts of the polymer-peptide block copolymers PS-E and K-PEG resulted in the formation of coiled-coil complexes between the peptides and subsequently in the formation of the amphiphilic triblock copolymer PS-E/K-PEG. Aqueous self-assembly of the separate peptides (E and K), the block copolymers (PS-E and K-PEG), and equimolar mixtures thereof was studied by circular dichroism, dynamic light scattering, and cryogenic transmission electron microscopy. It was found that the noncovalent PS-E/K-PEG copolymer assembled into rodlike micelles, while in all other cases, spherical micelles were observed. Temperature-dependent studies revealed the reversible nature of the coiled-coil complex and the influence of this on the morphology of the aggregate. A possible mechanism for these transitions based on the interfacial free energy and the free energy of the hydrophobic blocks is discussed. The self-assembly of the polymer-peptide conjugates is compared to that of polystyrene-b-poly(ethylene glycol), emphasizing the importance of the coiled-coil peptide block in determining micellar structure and dynamic behavior.

Uniting Polypeptides with Sequence-Designed Peptides: Synthesis and Assembly of Poly(γ-benzyl l-glutamate)-b-Coiled-Coil Peptide Copolymers

A new class of peptide has been created, polypeptide-b-designed peptides, which unites the useful qualities of the two constituent peptide types. We demonstrate the synthesis and self-assembly possibilities of this class of peptide chimera with a series of amphiphilic polypeptide-b-designed peptides in which the hydrophobic block is poly(gamma-benzyl l-glutamate) (PBLG) and the hydrophilic block is a coiled-coil forming peptide (denoted E). The synthetic approach was to synthesize the coiled-coil forming peptide on the solid phase, followed by the ring-opening polymerization of gamma-benzyl l-glutamate N-carboxyanhydride, initiated from the N-terminal amine of the peptide E on the solid support. The polypeptide-b-peptide was then cleaved from the resin, requiring no further purification. Peptide E contains 22 amino acids, while the average length of the PBLG block ranged from 36 to 250 residues. This new class of peptide was applied to create a modular system, which relied on juxtaposing the properties of the component peptide types, namely the broad size range and structure-inducing characteristics of the polypeptide PBLG blocks, and the complex functionality of the sequence-designed peptide. Specifically, the different PBLG block lengths could be connected noncovalently with various hydrophilic blocks via the specific coiled-coil folding of E with K or K-poly(ethylene glycol), where K is a peptide of complementary amino acid sequence to E. In this way, nanostructures could be formed in water at neutral pH over the entire compositional range, which has not been demonstrated previously with such large PBLG blocks. It was found that the size, morphology (polymersomes or bicelles), and surface functionality could be specified by combining the appropriate modular building blocks. The self-assembled structures were characterized by dynamic light scattering, circular dichroism, scanning electron microscopy, cryogenic-transmission electron microscopy, fluorescence spectroscopy, and zeta-potential measurements. Finally, as the structures are able to encapsulate water-soluble compounds, and the surfaces are easily functionalized via the coiled-coil binding, it is expected that these peptide-based nanocapsules will be able to act as delivery vehicles to specific targets in the body.

Polymer‐Peptide Block Copolymers – An Overview and Assessment of Synthesis Methods

Incorporating peptide blocks into block copolymers opens up new realms of bioactive or smart materials. Because there are such a variety of peptides, polymers, and hybrid architectures that can be imagined, there are many different routes available for the synthesis of these chimera molecules. This review summarizes the contemporary strategies in combining synthesis techniques to create well-defined peptide-polymer hybrids that retain the vital aspects of each disparate block. Living polymerization can be united with the molecular-level control afforded by peptide blocks to yield block copolymers that not only have precisely defined primary structures, but that also interact with other (bio)molecules in a well defined manner.

Rapid preparation of polymersomes by a water addition/solvent evaporation method

In this article we demonstrate a rapid water addition/solvent evaporation method to produce polymersomes with controllable sizes. For this method a solution of an amphiphilic block copolymer in THF is quickly mixed with an aqueous solution, followed by organic solvent evaporation under reduced pressure using a rotary evaporator. The parameters that influence the formation, size, and stability of the polymersomes are easily controlled, and the entire process can take less than five minutes. The method was initially tested with a series of rod–rod peptidic block copolymers, where the hydrophilic block is a charged designed peptide, and the hydrophobic block is poly(γ-benzyl L-glutamate) with varying degrees of polymerization (35–250 monomers), and the polymersome formation was monitored and confirmed with dynamic light scattering, optical microscopy, and transmission electron microscopy. The widespread applicability of the technique was also proven with more traditional charged and non-charged coil–coil block copolymers of varying length. The method was found to be very robust with regards to salt concentration and initial mixing, and the polymersome size could be precisely adjusted over a wide range, with the same block copolymer forming polymersomes ranging from ∼200 nm to ∼2 µm in diameter. Given its simplicity, versatility, and speed, the water addition/solvent evaporation method described here is a very practical tool for polymersome preparation.

Detergent-Aided Polymersome Preparation

Until now, most preparative methods used to form polymeric vesicles involve either organic cosolvents or sonication. In this communication, we demonstrate for the first time a detergent-aided method to produce polymersomes. Peptidic polymersomes were formed from the rod-rod block copolymer PBLG(36)-E, where PBLG is hydrophobic poly(gamma-benzyl l-glutamate) and E is a hydrophilic designed peptide. The block copolymer was first solubilized by detergent micelles in aqueous buffer, after which the concentration of detergent was reduced by dilution, transforming the particle morphology in solution from mixed micelles to polymersomes. The polymersome formation was monitored with dynamic light scattering and confirmed with transmission electron microscopy. Polymersomes with average diameters of approximately 300 nm were obtained as well as discs with average diameters of approximately 100 nm. This detergent-based method can be used to create polymersomes with a range of properties, as verified by its application to another biocompatible block copolymer, the flexible polybutadiene(46)-b-poly(ethylene glycol)(30). The technique will be particularly useful when delicate biomacromolecules such as (membrane) proteins, peptides, or nucleic acids are to be encapsulated in the polymersomes because the detergents used are compatible with these compounds, and the possible denaturing effect of sonication or organic solvents on the biological activity of the molecule of interest is avoided.

Controlled liposome fusion mediated by SNARE protein mimics

The fusion of lipid membranes is essential for the delivery of chemicals across biological barriers to specific cellular locations. Intracellular membrane fusion is particularly precise and is critically mediated by SNARE proteins. To allow membrane fusion to be better understood and harnessed we have mimicked this important process with a simple bottom-up model in which synthetic fusogens replicate the essential features of SNARE proteins. In our fusogens, the coiled-coil molecular recognition motif of SNARE proteins is replaced by the coiled-coil E/K peptide complex, which is one-ninth the size. The peptides are anchored in liposome membranes via pegylated lipids. Here we discuss how the liposome fusion process is controlled by different parameters within the minimal model. The lipopeptide fusogens form specific coiled coils that dock liposomes together, resulting in the merging of membranes via the stalk intermediate. Unusually for model systems, the lipopeptides can rapidly lead to fusion of entire liposome populations and the liposomes can undergo many rounds of fusion. The rate and extent of fusion and the number of fusion rounds can be manipulated by adjusting the fusogen and liposome concentrations. For example, these parameters can be tuned such that tens of thousands of ∼100 nm liposomes fuse into a single giant liposome ∼10 μm in diameter; alternatively, conditions can be selected such that only two liposomes fuse. The improved understanding of membrane fusion shows how application-specific fusion attributes can be achieved, and paves the way for controlled nanoreactor mixing and controlled delivery of cargo to cells.

Introducing Quadrupole Interactions into the Peptide Design Toolkit

Proteins and peptides are often described as the machinery of life. Even the simplest bacteria have hundreds of proteins, and these proteins work in concert with each other to conduct thousands of distinct functions. The fidelity of these functions relies on the specificity of interactions between the units of the machinery, between the proteins. By studying the forms and functions of proteins, and tracing these back to amino acid sequences the “rules” for their self-assembly can be obtained, thus allowing de novo peptide design and yielding novel forms and functions. The design of linear amino acid sequences that fold into defined secondary structures, or motifs, such as a-helices or b-sheets is relatively well understood. The greater challenge is to engineer specific interactions between these motifs, that is between precisely positioned side chains. An improved ability to direct intermolecular interactions will increase the functionality of the peptide assemblies that we are able to construct, and correspondingly, increase their potential applications. Until recently, the chemical toolkit for introducing peptide–peptide molecular recognition has consisted of four tools, or noncovalent interactions between amino acid side chains. These are ionic interactions, hydrogen bonding, hydrophobic interactions, and p stacking. Zheng and Gao have recently described a new tool, the quadrupole interaction (Figure 1), which is a refinement of p stacking. The ways in which these tools are utilized to impart specificity to peptide interactions are touched upon in the following paragraphs, with particular reference to coiled coils, which are the best understood peptide machinery. 5] 1. Hydrophobic interactions. The hydrophobic effect is generally the strongest component in protein and peptide quaternary interactions, and the degree of steric matching between hydrophobic side chains is important for increasing the stability of the complexes. However the interactions are not as specific as those between hydrophilic side chains. With regards to coiled coils, hydrophobic design principles have been used to greatest effect in determining the oligomerization state or relative orientation of ahelices in complexes rather than specifying binding partners. This strong but non-exclusive form of molecular recognition is complemented with other binding mechanisms in de novo peptides. 2. Ionic interactions. Amino acids with charged side chains are important determinants of specificity in peptide complexes, and this is the most frequently used strategy. Specificity is often achieved by negative design, whereby a unique structure is achieved by destabilizing the other possible complexes. Many heterodimeric coiled coils have charged residues bordering the hydrophobic core such that one helix is positively charged and the other negatively charged, hence preventing homodimeric coiledcoils from forming. Controlling intermolecular electrostatic interactions by using changes in pH values or salt concentrations can also be used to switch peptide binding specificity. This concept has been demonstrated with iterative pH cycles, specifically replacing one, two, or all three initial helices of a coiled-coil trimer. 3. Hydrogen bonds. Hydrogen bonds are also used to impart specificity to coiled coils by the placement of hydrogenFigure 1. Space-filling model of the aromatic side chains of phenylalanine and perfluorophenylalanine stacked face-to-face. The distribution of electrostatic potential is indicated, with blue being positive, and red negative. Phenyl and perfluorophenyl have opposite quadrupole moments, allowing a net electrostatic attraction between the p faces to occur.

Influence of pegylation on peptide-mediated liposome fusion

The effect of surface-attached PEG on the peptide-mediated fusion of liposomes was investigated. A complementary pair of coiled-coil forming lipidated peptides was introduced to two batches of small unilamellar liposomes separately. Upon mixing, efficient liposome membrane fusion was apparent when the liposomes were not decorated with pegylated lipids, however when the liposomes were pegylated the fusion was inhibited. A FRET-based fluorescence assay indicated that the fusion can be prevented effectively with less than two mole percent of pegylated lipid. DLS and CD spectroscopy were used to further evaluate the influence of pegylation on fusion. These data revealed that the pegylated lipids inhibit peptide complex formation and liposome docking, thereby preventing liposome fusion at the initial stage of the process. In contrast, when the PEG is not covalently attached to the liposome, no fusion inhibition was observed. Thus we conclude that the steric effect of the surface-bound PEG chains, which prevents sustained docking of liposomes, is the main cause of fusion inhibition.