In JoVE (1)

Other Publications (28)

Articles by Adam W. Perriman in JoVE

 JoVE Bioengineering

Synthesis of Cationized Magnetoferritin for Ultra-fast Magnetization of Cells

1Bristol Centre for Functional Nanomaterials, University of Bristol, 2Department of Materials, Imperial College London, 3Self Assembly Group, CIC nanoGUNE, 4Ikebasque, Basque Foundation for Science, 5School of Cellular and Molecular Medicine, University of Bristol, 6H.H. Wills Physics Laboratory, University of Bristol

JoVE 54785

Other articles by Adam W. Perriman on PubMed

Protein-poly(silicic) Acid Interactions at the Air/solution Interface

The Journal of Physical Chemistry. B. Nov, 2005  |  Pubmed ID: 16853707

The structure of the interface generated by a spread layer of beta-casein on an aqueous colloidal poly(silicic) acid subphase is described. The results are compared with data for the protein alone spread at the air/water interface and the silicate solution. Films develop at the air-solution interface and a strong pH dependence of the interaction causing this is demonstrated. Reflectometry with X-rays and neutrons was used to probe the interaction as a function of subphase pH and film compression. Film thickness, tau/A, scattering length density, rho/A(-2), water volume fraction, phi(w), and surface coverage, Gamma/mg m(-2), were used to quantify the interfacial structure. Where possible, the X-ray and neutron data sets were co-refined enabling phi(w) to be evaluated without assumption regarding the protein density. At pH 5-7, strong protein-silicate interaction occurred, the interface comprising three regions: a discrete protein upper layer on top of a 15 +/- 2 A layer of silicated material followed by a diffuse layer that extended into the subphase.

Effect of the Air-water Interface on the Stability of Beta-lactoglobulin

The Journal of Physical Chemistry. B. Dec, 2007  |  Pubmed ID: 17994721

We report the X-ray and neutron reflectometry measurements of the structural changes caused by chemical denaturation of a surface excess of the bovine milk protein, beta-lactoglobulin. The thickness of the diffuse protein surface layer was used as an order parameter as there was no corresponding increase in the surface excess as a function of guanidinium chloride (G.HCl) concentration. A thermodynamic analysis performed gave the interfacial free energy of unfolding in the absence of a denaturant (DeltaG(0)). This energy, lower than the free energy of unfolding bulk solution, shows that the air-water interface has a destabilizing effect on protein structure up to 50 kJ mol(-1).

Surface Movement in Water of Splendipherin, the Aquatic Male Sex Pheromone of the Tree Frog Litoria Splendida

The FEBS Journal. Jul, 2008  |  Pubmed ID: 18494800

The aquatic sex pheromone splendipherin (GLVSSIGKALGGLLADVVKSKGQPA-OH) of the male green tree frog Litoria splendida moves across the surface of water to reach the female. Surface pressure and X-ray reflectometry measurements confirm that splendipherin is a surface-active molecule, and are consistent with it having an ordered structure, whereby the hydrophilic portion of the peptide interacts with the underlying water and the hydrophobic region is adjacent to the vapour phase. The movement of splendipherin over the surface of water is caused by a surface pressure gradient. In order to better define the structure of splendipherin at the water/air interface we used 2D NMR studies of the pheromone with the solvent system trifluoroethanol/water (1 : 1 v/v). In this solvent system, splendipherin adopts a bent alpha helix from residues V3 to K21. The bending of the helix occurs in the centre of the peptide in the vicinity of G11 and G12. The region of splendipherin from V3 to G11 has well-defined amphipathicity, whereas the amphipathicity from G12 to A25 is reduced by K19 and P24 intruding into the hydrophobic and hydrophilic regions respectively. A helical structure is consistent with X-ray reflectometry data.

Effect of the Air-water Interface on the Structure of Lysozyme in the Presence of Guanidinium Chloride

The Journal of Physical Chemistry. B. Aug, 2008  |  Pubmed ID: 18616315

We report observations of the changes in the surface structure of lysozyme adsorbed at the air-water interface produced by the chemical denaturant guanidinium chloride. A primary result is the durability of the adsorbed surface layer to denaturation, as compared to the molecule in the bulk solution. Data on the surface film were obtained from X-ray and neutron reflectivity measurements and modeled simultaneously. The behavior of lysozyme in G.HCl solutions was determined by small-angle X-ray scattering. For the air-water interface, determination of the adsorbed protein layer dimensions shows that at low to moderate denaturant concentrations (up to 2 mol L(-1)), there is no significant distortion of the protein's tertiary structure at the interface, as changes in the orientation of the protein are sufficient to model data. At higher denaturant concentrations, time-dependent multilayer formation occurred, indicating molecular aggregation at the surface. Methodologies to predict the protein orientation at the interface, based on amino acid residues' surface affinities and charge, were critiqued and validated against our experimental data.

Solvent-free Protein Liquids and Liquid Crystals

Angewandte Chemie (International Ed. in English). 2009  |  Pubmed ID: 19603445

Synthetic Viruslike Particles and Hybrid Constructs Based on Lipopeptide Self-assembly

Small (Weinheim an Der Bergstrasse, Germany). Jun, 2010  |  Pubmed ID: 20461723

Reversible Dioxygen Binding in Solvent-free Liquid Myoglobin

Nature Chemistry. Aug, 2010  |  Pubmed ID: 20651722

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

Chembiochem : a European Journal of Chemical Biology. Jan, 2011  |  Pubmed ID: 21132689

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.

Liquid Proteins--a New Frontier for Biomolecule-based Nanoscience

ACS Nano. Aug, 2011  |  Pubmed ID: 21749034

Solid, liquid, or gas? The physical states adopted by nano-objects such as proteins are critically dependent on the size and range of the intermolecular attractive forces, and as a consequence, pure liquids comprising solventless melts of structurally intact, discrete functional nanoconstructs are conspicuously absent. Here we describe how globular proteins can be surface-engineered such that anhydrous powders of these nanoscale objects melt at close to room temperature to produce solvent-free liquids comprising exceedingly high concentrations of structurally and functionally intact biomolecules. These liquids offer unprecedented opportunities in bionanomaterials research, represent a new phase for protein chemistry, and challenge the existing paradigm on the role of water molecules in protein folding and function.

Peptide-nucleotide Microdroplets As a Step Towards a Membrane-free Protocell Model

Nature Chemistry. Aug, 2011  |  Pubmed ID: 21860462

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.

Nematic Director-induced Switching of Assemblies of Hexagonally Packed Gold Nanorods

Advanced Materials (Deerfield Beach, Fla.). Aug, 2012  |  Pubmed ID: 22761047

Self-assembled disc-shaped clusters of hexagonally packed, thiol-functionalized gold nanorods are prepared and dispersed in thermotropic nematic liquid crystals. The resultant hybrid complex fluids exhibit colloidal anisotropy with very high orientational order and are characterized by SAXS as shown in the figure. Precise, reconfigurable control of the cluster orientation at very low electric field strengths (0.18 V μm(-1) ) is achieved.

Liquid Viruses by Nanoscale Engineering of Capsid Surfaces

Advanced Materials (Deerfield Beach, Fla.). Aug, 2012  |  Pubmed ID: 22807140

Surface engineering of plant virus capsids via cationization (1) and stoichiometric coupling of a polymer surfactant coronal layer (2) produces a highly concentrated, solvent-free liquid virus at 28 °C. These ionic bionanoconstructs are viscoelastic, retain plant infectivity and can be dispersed in a range of organic solvents for aerosol delivery.

A Polymer Surfactant Corona Dynamically Replaces Water in Solvent-free Protein Liquids and Ensures Macromolecular Flexibility and Activity

Journal of the American Chemical Society. Aug, 2012  |  Pubmed ID: 22853639

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.

Magnetizing DNA and Proteins Using Responsive Surfactants

Advanced Materials (Deerfield Beach, Fla.). Dec, 2012  |  Pubmed ID: 22945776

DNA chains and their movement in solvent may now be controlled simply by surfactant binding and the switching "on" and "off" of a magnetic field adding a new paradigm to the study and control, condensation and manipulation of DNA (and other biomolecules). Such control is essential for biotechnological applications such as transfection and the regulation of gene suppression, as well as in materials science concerning soft molecular self-assemblies.

Enzymatically Active Self-standing Protein-polymer Surfactant Films Prepared by Hierarchical Self-assembly

Advanced Materials (Deerfield Beach, Fla.). Apr, 2013  |  Pubmed ID: 23381887

Cross-linked protein-polymer surfactant films consisting of enzymatically active hybrid nanoclusters are prepared using a novel approach based on electrostatically mediated hierarchical self-assembly. The free-standing films are structurally robust, highly hydrophilic, and exhibit sustained fluorescence or recyclable enzymatic phosphatase or oxido-reductase behavior.

Isolation of a Highly Reactive β-sheet-rich Intermediate of Lysozyme in a Solvent-free Liquid Phase

The Journal of Physical Chemistry. B. Jul, 2013  |  Pubmed ID: 23790147

The thermal denaturation of solvent-free liquid lysozyme at temperatures in excess of 200 °C was studied by synchrotron radiation circular dichroism spectroscopy. Temperature-dependent changes in the secondary structure were used to map the equilibrium denaturation pathway and characterize a reactive β-sheet-rich unfolding intermediate that was stable in the solvent-free liquid phase under anhydrous conditions but which underwent irreversible aggregation in the presence of water. The unfolding intermediate had a transition temperature of 78 °C and was extremely stable to temperature, eventually reaching the fully denatured state at 178 °C. We propose that the three-stage denaturation pathway arises from the decreased stability of the native state due to the absence of any appreciable hydrophobic effect, along with an entropically derived stabilization of the reactive intermediate associated with molecular crowding in the solvent-free liquid.

Enhanced Catalytic Activity in Organic Solvents Using Molecularly Dispersed Haemoglobin-polymer Surfactant Constructs

Chemical Communications (Cambridge, England). Oct, 2013  |  Pubmed ID: 24018483

The surface of haemoglobin (Hb) is chemically modified to produce molecular dispersions of discrete core-shell Hb-polymer surfactant bionanoconjugates in water and organic solvents. The hybrid nanoconstructs exhibit peroxidase-like catalytic activity with enhanced turnover rates compared with native Hb in water.

Redox Transitions in an Electrolyte-free Myoglobin Fluid

Journal of the American Chemical Society. Dec, 2013  |  Pubmed ID: 24245589

Redox responses associated with the heme prosthetic group in a myoglobin-polymer surfactant solvent-free liquid are investigated for the first time in the absence of an electrolyte solution. Cyclic voltammograms from the biofluid exhibit responses that are consistent with planar diffusion of mobile charges in the melt. Temperature-dependent dynamic electrochemical and rheological responses are rationalized in terms of the effective electron hopping rate between heme centers and the transport of intrinsic ionic species in the viscous protein liquid.

Fatty Acid Membrane Assembly on Coacervate Microdroplets As a Step Towards a Hybrid Protocell Model

Nature Chemistry. Jun, 2014  |  Pubmed ID: 24848239

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.

Self-organization of Glucose Oxidase-polymer Surfactant Nanoconstructs in Solvent-free Soft Solids and Liquids

The Journal of Physical Chemistry. B. Oct, 2014  |  Pubmed ID: 25201462

An anisotropic glucose oxidase-polymer surfactant nanoconjugate is synthesized and shown to exhibit complex temperature-dependent phase behavior in the solvent-free state. At close to room temperature, the nanoconjugate crystallizes as a mesolamellar soft solid with an expanded interlayer spacing of ca. 12 nm and interchain correlation lengths consistent with alkyl tail-tail and PEO-PEO ordering. The soft solid displays a birefringent spherulitic texture and melts at 40 °C to produce a solvent-free liquid protein without loss of enzyme secondary structure. The nanoconjugate melt exhibits a birefringent dendritic texture below the conformation transition temperature (Tc) of glucose oxidase (58 °C) and retains interchain PEO-PEO ordering. Our results indicate that the shape anisotropy of the protein-polymer surfactant globular building block plays a key role in directing mesolamellar formation in the solvent-free solid and suggests that the microstructure observed in the solvent-free liquid protein below Tc is associated with restrictions in the intramolecular motions of the protein core of the nanoconjugate.

Enzyme Activity in Liquid Lipase Melts As a Step Towards Solvent-free Biology at 150 °C

Nature Communications. Oct, 2014  |  Pubmed ID: 25284507

Water molecules play a number of critical roles in enzyme catalysis, including mass transfer of substrates and products, nucleophilicity and proton transfer at the active site, and solvent shell-mediated dynamics for accessing catalytically competent conformations. The pervasiveness of water in enzymolysis therefore raises the question concerning whether biocatalysis can be undertaken in the absence of a protein hydration shell. Lipase-mediated catalysis has been undertaken with reagent-based solvents and lyophilized powders, but there are no examples of molecularly dispersed enzymes that catalyse reactions at sub-solvation levels within solvent-free melts. Here we describe the synthesis, properties and enzyme activity of self-contained reactive biofluids based on solvent-free melts of lipase-polymer surfactant nanoconjugates. Desiccated substrates in liquid (p-nitrophenyl butyrate) or solid (p-nitrophenyl palmitate) form can be mixed or solubilized, respectively, into the enzyme biofluids, and hydrolysed in the solvent-free state. Significantly, the efficiency of product formation increases as the temperature is raised to 150 °C.

Molecular Dynamics Simulations Reveal a Dielectric-responsive Coronal Structure in Protein-polymer Surfactant Hybrid Nanoconstructs

Journal of the American Chemical Society. Dec, 2014  |  Pubmed ID: 25380317

Solvent-free liquid proteins are a new class of thermally stable hybrid bionanomaterials that are produced by extensive lyophilization of aqueous solutions of protein-polymer surfactant nanoconjugates followed by thermal annealing. The hybrid constructs, which consist of a globular protein core surrounded by a monolayer of electrostatically coupled polymer surfactant molecules, exhibit nativelike structure, function, and backbone dynamics over a large temperature range. Despite the key importance of the polymer surfactant shell, very little is known about the atomistic structure of the corona and how it influences the phase behavior and properties of these novel nanoscale objects. Here we present molecular dynamics simulations of protein-polymer surfactant nanoconjugates consisting of globular cores of myoglobin or lysozyme and demonstrate that the derived structural parameters are highly consistent with experimental values. We show that the coronal layer structure is responsive to the dielectric constant of the medium and that the mobility of the polymer surfactant molecules is significantly hindered in the solvent-free state, providing a basis for the origins of retained protein dynamics in these novel biofluids. Taken together, our results suggest that the extension of molecular dynamics simulations to hybrid nanoscale objects could be of generic value in diverse areas of soft matter chemistry, bioinspired engineering, and biomolecular nanotechnology.

Photocatalytic Multiphase Micro-droplet Reactors Based on Complex Coacervation

Chemical Communications (Cambridge, England). May, 2015  |  Pubmed ID: 25896224

We describe the synthesis and characterisation of novel photocatalytic multiphase micro-droplet reactors comprising TiO2 nanosheets dispersed in poly(diallyldimethylammonium) chloride and adenosine 5'-triphosphate or poly(ethylene glycol) 4-nonylphenyl 3-sulfopropyl complex coacervates. We demonstrate significant variations in the degree of equilibrium partitioning of small molecule dyes into the coacervate droplet systems and exploit this behaviour to successfully conduct selective photocatalytic dye degradation.

Artificial Membrane-binding Proteins Stimulate Oxygenation of Stem Cells During Engineering of Large Cartilage Tissue

Nature Communications. Jun, 2015  |  Pubmed ID: 26080734

Restricted oxygen diffusion can result in central cell necrosis in engineered tissue, a problem that is exacerbated when engineering large tissue constructs for clinical application. Here we show that pre-treating human mesenchymal stem cells (hMSCs) with synthetic membrane-active myoglobin-polymer-surfactant complexes can provide a reservoir of oxygen capable of alleviating necrosis at the centre of hyaline cartilage. This is achieved through the development of a new cell functionalization methodology based on polymer-surfactant conjugation, which allows the delivery of functional proteins to the hMSC membrane. This new approach circumvents the need for cell surface engineering using protein chimerization or genetic transfection, and we demonstrate that the surface-modified hMSCs retain their ability to proliferate and to undergo multilineage differentiation. The functionalization technology is facile, versatile and non-disruptive, and in addition to tissue oxygenation, it should have far-reaching application in a host of tissue engineering and cell-based therapies.

Dynamic Behavior in Enzyme-Polymer Surfactant Hydrogel Films

Advanced Materials (Deerfield Beach, Fla.). Feb, 2016  |  Pubmed ID: 26676924

Dynamic protein-polymer surfactant films are highly hydrophilic and show a soft solid to hydrogel transition upon hydration to produce a swollen hydrogel. An unusual reversible autospreading/self-folding response is observed when the water-saturated films are transferred from water into air.

3D Bioprinting Using a Templated Porous Bioink

Advanced Healthcare Materials. Jul, 2016  |  Pubmed ID: 27125336

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.

Strategies for Cell Membrane Functionalization

Experimental Biology and Medicine (Maywood, N.J.). May, 2016  |  Pubmed ID: 27229904

The ability to rationally manipulate and augment the cytoplasmic membrane can be used to overcome many of the challenges faced by conventional cellular therapies and provide innovative opportunities when combined with new biotechnologies. The focus of this review is on emerging strategies used in cell functionalization, highlighting both pioneering approaches and recent developments. These will be discussed within the context of future directions in this rapidly evolving field.

Effect of Bioconjugation on the Reduction Potential of Heme Proteins

Biomacromolecules. Oct, 2016  |  Pubmed ID: 27650815

The modification of protein surfaces employing cationic and anionic species enables the assembly of these biomaterials into highly sophisticated hierarchical structures. Such modifications can allow bioconjugates to retain or amplify their functionalities under conditions in which their native structure would be severely compromised. In this work, we assess the effect of this type of bioconjugation on the redox properties of two model heme proteins, that is, cytochrome c (CytC) and myoglobin (Mb). In particular, the work focuses on the sequential modification by 3-dimethylamino propylamine (DMAPA) and 4-nonylphenyl 3-sulfopropyl ether (S1) anionic surfactant. Bioconjugation with DMAPA and S1 are the initial steps in the generation of pure liquid proteins, which remain active in the absence of water and up to temperatures above 150 °C. Thin-layer spectroelectrochemistry reveals that DMAPA cationization leads to a distribution of bioconjugate structures featuring reduction potentials shifted up to 380 mV more negative than the native proteins. Analysis based on circular dichroism, MALDI-TOF mass spectrometry, and zeta potential measurements suggest that the shift in the reduction potentials are not linked to protein denaturation, but to changes in the spin state of the heme. These alterations of the spin states originate from subtle structural changes induced by DMAPA attachment. Interestingly, electrostatic coupling of anionic surfactant S1 shifts the reduction potential closer to that of the native protein, demonstrating that the modifications of the heme electronic configuration are linked to surface charges.

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