Adam W. Perriman

Peptide–nucleotide microdroplets as a step towards a membrane-free protocell model

Although phospholipid bilayers are ubiquitous in modern cells, their impermeability, lack of dynamic properties, and synthetic complexity are difficult to reconcile with plausible pathways of proto-metabolism, growth and division. Here, we present an alternative membrane-free model, which demonstrates that low-molecular-weight mononucleotides and simple cationic peptides spontaneously accumulate in water into microdroplets that are stable to changes in temperature and salt concentration, undergo pH-induced cycles of growth and decay, and promote α-helical peptide secondary structure. Moreover, the microdroplets selectively sequester porphyrins, inorganic nanoparticles and enzymes to generate supramolecular stacked arrays of light-harvesting molecules, nanoparticle-mediated oxidase activity, and enhanced rates of glucose phosphorylation, respectively. Taken together, our results suggest that peptide-nucleotide microdroplets can be considered as a new type of protocell model that could be used to develop novel bioreactors, primitive artificial cells and plausible pathways to prebiotic organization before the emergence of lipid-based compartmentalization on the early Earth.

Fatty acid membrane assembly on coacervate microdroplets as a step towards a hybrid protocell model

Mechanisms of prebiotic compartmentalization are central to providing insights into how protocellular systems emerged on the early Earth. Protocell models are based predominantly on the membrane self-assembly of fatty-acid vesicles, although membrane-free scenarios that involve liquid-liquid microphase separation (coacervation) have also been considered. Here we integrate these alternative models of prebiotic compartmentalization and develop a hybrid protocell model based on the spontaneous self-assembly of a continuous fatty-acid membrane at the surface of preformed coacervate microdroplets prepared from cationic peptides/polyelectrolytes and adenosine triphosphate or oligo/polyribonucleotides. We show that the coacervate-supported membrane is multilamellar, and mediates the selective uptake or exclusion of small and large molecules. The coacervate interior can be disassembled without loss of membrane integrity, and fusion and growth of the hybrid protocells can be induced under conditions of high ionic strength. Our results highlight how notions of membrane-mediated compartmentalization, chemical enrichment and internalized structuration can be integrated in protocell models via simple chemical and physical processes.

Solvent‐Free Protein Liquids and Liquid Crystals

Nanostructures such as functionalized nanoparticles and superlattices have wide-ranging applications in diverse areas. Although these materials are invariably used in the form of aqueous/organic dispersions, ultrathin films, or bulk powders, Giannellis and co-workers have recently pioneered an approach to preparing functionalized inorganic nanostructures with liquidlike behavior. These are produced by electrostatically grafting an organic canopy layer onto the surface of charged nanoparticles of silica, iron oxides, and titania for example, to provide a fluidization medium for the preparation of solvent-free nanoparticle ionic fluids. Like nanoscale objects in general, proteins exhibit persistent structures with dimensions that exceed the range of their intermolecular forces, such that liquid–vapor co-existence is unattainable. As a consequence, solid-state proteins sublime at low pressures or thermally degrade under ambient conditions: thus, there are no known liquid proteins in the absence of solvent. Herein, we report, to our knowledge, the first example of a solvent-free liquid protein. Specifically, we report the preparation and properties of a protein melt based on a stoichiometric ferritin–polymer nanoscale construct with surface modifications that extend the range of intermolecular interactions to a length scale that is commensurate with fluidity in the absence of water. Moreover, we show that these spherically shaped nano-constructs undergo anisotropic ordering during melting at 30 8C to produce a viscoelastic protein liquid that exhibits thermotropic liquid-crystalline behavior, and which subsequently transforms to a Newtonian fluid at temperatures above 40 8C and is stable up to a temperature of 405 8C. The method, which utilizes the sitespecificity of surface amino acid residues and high degree of uniformity in ferritin molecular architecture to produce discrete single-component ferritin–polymer constructs (Supporting Information, Figure S1), should be readily accessible to exploitation as a facile route to solvent-free liquid proteins and enzymes in general. Electrostatically induced complexation of cationized ferritin (C-Fn), comprising approximately 240 covalently coupled N,N-dimethyl-1,3-propanediamine (DMPA) groups per molecule (10 DMPA per subunit; Supporting Information, Figure S2), with the anionic polymer surfactant C9H19C6H4-(OCH2CH2)20O(CH2)3SO3 (S) resulted in the formation of the ionic nanoconstruct [C-Fn][S]. Sedimentationcoefficient distributions obtained by analytical ultracentrifugation of extensively dialyzed aqueous solutions of [C-Fn][S] showed a single peak centered at 37 S compared with values of 51 or 0.5 S for C-Fn or S alone (Supporting Information, Figure S3). The decrease in density of the conjugate compared with non-complexed C-Fn, as well as the absence of unbound surfactant, were consistent with a discrete protein– polymer ionic construct. Significantly, calculations based on comparative density variations coupled with changes in molecular radius associated with electrostatic conjugation, gave a [C-Fn]:[S] stoichiometry of 1:264 (Supporting Information). This was consistent with a persistent surface chargematched ferritin–polymer nanostructure comprising on average eleven electrostatically coupled polymer chains per cationized subunit and a theoretical polymer content of 34 wt%. Similar experiments and calculations undertaken on conjugates prepared from native ferritin also confirmed the formation of a single component construct but with a [Fn]:[S] stoichiometry of 1:96 (i.e. four surfactant molecules per subunit). Protein melts were prepared in the absence of water by lyophilization of the aqueous [C-Fn][S] solutions to produce a low-density solid that was subsequently annealed at 50 8C to produce a transparent, viscous, red liquid that remained fluid when cooled to room temperature, but re-solidified at 50 8C (Figure 1). TEM studies of the melt revealed discrete electron-dense nanoparticles, approximately 8 nm in diameter, indicating that the protein nanostructure remained structurally intact in the liquid state (Supporting Information, Figure S4). The melts were readily soluble in water or dichloromethane. Thermogravimetric analysis of the melt gave a water content of less than 2% and a residual mass at 1200 8C of 13%, which corresponded to the iron oxide cores of the protein. The melt decomposed at 405 8C, compared with 315 and 380 8C for native ferritin and S, respectively, indicating cooperative stabilization and decomposition that were consistent with a stoichiometric complex. Significantly, differential scanning calorimetry (DSC) on annealed samples showed significant changes in the enthalpic phase transitions between S and the [C-Fn][S] ionic nanoconstruct (Figure 2a). For S, a large asymmetric endothermic transition consisting of a shoulder at 20 8C and a minimum at 27.5 8C was evident on heating, consistent with a two-stage process involving melting [*] Dr. A. W. Perriman, Prof. S. Mann Centre for Organized Matter Chemistry, School of Chemistry University of Bristol, Bristol BS8 1TS (UK) E-mail: s.mann@bristol.ac.uk

Preparation of high quality nanowires by tobacco mosaic virus templating of gold nanoparticles

A protocol for the preparation of stable suspensions of well-defined metallized tobacco mosaic virus (TMV) nanorods in high yields and with uniform coatings is reported. Inorganic growth on the virion surface was controlled by adding aliquots of gold precursor (HAuCl4) and reducing agent (NaBH4) incrementally over five addition–reduction cycles to produce hybrid anisotropic nanostructures consisting of densely packed gold nanoparticles. Several novel steps, including addition of ethanol after the first addition–reduction cycle and wrapping of the TMV/Au nanohybrids with poly-L-lysine, were introduced into the protocol to ensure high homogeneity and stability in the nanowire suspension. Control of the interparticle spacing in the metallized nanostructures was achieved by restricting the number of reaction cycles used to less than four iterations. The importance of bio-mediated reduction was probed using laser light scattering microscopy. The results indicated that short incubation times were consistent with increased nanoparticle monodispersity and high fidelity replication.

Reversible dioxygen binding in solvent-free liquid myoglobin.

The ensemble of forces that stabilize protein structure and facilitate biological function are intimately linked with the ubiquitous aqueous environment of living systems. As a consequence, biomolecular activity is highly sensitive to the interplay of solvent-protein interactions, and deviation from the native conditions, for example by exposure to increased thermal energy or severe dehydration, results in denaturation and subsequent loss of function. Although certain enzymes can be extracted into non-aqueous solvents without significant loss of activity, there are no known examples of solvent-less (molten) liquids of functional metalloproteins. Here we describe the synthesis and properties of room-temperature solvent-free myoglobin liquids with near-native structure and reversible dioxygen binding ability equivalent to the haem protein under physiological conditions. The realization of room-temperature solvent-free myoglobin liquids with retained function presents novel challenges to existing theories on the role of solvent molecules in structural biology, and should offer new opportunities in protein-based nanoscience and bionanotechnology.

Engineered synthetic virus-like particles and their use in vaccine delivery

Engineered nanoparticles have been designed based on the self‐assembling properties of synthetic coiled‐coil lipopeptide building blocks. The presence of an isoleucine zipper within the lipopeptide together with the aggregating effects of an N‐terminal lipid drives formation of 20–25 nm nanoparticles in solution. Biophysical studies support a model in which the lipid is buried in the centre of the nanoparticle, with 20–30 trimeric helical coiled‐coil bundles radiating out into solution. A promiscuous T‐helper epitope and a synthetic B‐cell epitope mimetic derived from the circumsporozoite protein of Plasmodium falciparum have been linked to each lipopeptide chain, with the result that 60–90 copies of each antigen are displayed over the surface of the nanoparticle. These nanoparticles elicit strong humoral immune responses in mice and rabbits, including antibodies able to cross‐react with the parasite, thereby, supporting the potential value of this delivery system in synthetic vaccine design.

Polymer/nucleotide droplets as bio-inspired functional micro-compartments

Using a range of physical methods, we describe the formation, structure, stability, physical properties and uptake behavior of condensed liquid micro-droplets prepared by electrostatically induced complexation of poly(diallyldimethylammonium) chloride (PDDA) and adenosine triphosphate (ATP) in water. Depending on the PDDA monomer: ATP molar ratio, positively charged or charge-neutral droplets are produced spontaneously by simple mixing. The former are typically 60–600 nm in mean size and stable with respect to sedimentation up to temperatures of 85 °C, whilst the latter grow into droplets several tens of micrometres in diameter that coalesce into a macroscopic coacervate phase. Coacervation is inhibited at pH values less than 3 and at high ionic strength, confirming the importance of charge interactions in droplet formation and stability. The droplet interior is structurally homogeneous with no surrounding membrane, comprises dynamically fluctuating domains of partially desolvated polymer/nucleotide complexes, and has a dielectric constant considerably lower than water. As a consequence, dye molecules, porphyrin macrocycles, inorganic nanoparticles or globular proteins can be sequestered from the external water phase into the droplets to produce PDDA/ATP droplets comprising supramolecular J-aggregate nanostructures, magnetically responsive deformable fluids, or soft compartments with potential storage and release properties.

3D Bioprinting Using a Templated Porous Bioink

3D tissue printing with adult stem cells is reported. A novel cell-containing multicomponent bioink is used in a two-step 3D printing process to engineer bone and cartilage architectures.

Structure and function of the silicifying peptide R5

The 19-mer synthetic peptide known as R5 has been used widely in studies of peptide-driven silica condensation. Despite this, the structure and function of R5 have not yet been fully characterized. Here, we present a systematic study of R5 silicification focusing on three key variables: the concentration of the peptide, the concentration of the silica precursor silicic acid, and the solution pH. Additionally, we present the first study of R5 secondary structure in the presence and absence of silicic acid and introduce one-dimensional and two-dimensional solution NMR to probe both structure and higher-order peptide aggregation. We find that R5-directed silicification is linear with regard to silicic acid and H+ but, unexpectedly, that silicification appears to be cooperative with respect to peptide concentration. We also find that R5 is a random coil ensemble at subsaturating silicic acid concentrations and does not spontaneously self-assemble to form discrete aggregates in solution. These data contradict a model that invokes the functional micellization of R5 and provide a framework for future studies with the R5 peptide.

A polymer surfactant corona dynamically replaces water in solvent-free protein liquids and ensures macromolecular flexibility and activity.

The observation of biological activity in solvent-free protein-polymer surfactant hybrids challenges the view of aqueous and nonaqueous solvents being unique promoters of protein dynamics linked to function. Here, we combine elastic incoherent neutron scattering and specific deuterium labeling to separately study protein and polymer motions in solvent-free hybrids. Myoglobin motions within the hybrid are found to closely resemble those of a hydrated protein, and motions of the polymer surfactant coating are similar to those of the hydration water, leading to the conclusion that the polymer surfactant coating plasticizes protein structures in a way similar to hydration water.