The first well‐controlled aqueous atom‐transfer radical polymerization (ATRP) conducted in the open air is reported. This air‐tolerant ATRP was enabled by the continuous conversion of oxygen to ...carbon dioxide catalyzed by glucose oxidase (GOx), in the presence of glucose and sodium pyruvate as sequential sacrificial substrates. Controlled polymerization using initiators for continuous activator regeneration (ICAR) ATRP of oligo(ethylene oxide) methyl ether methacrylate (OEOMA, Mn=500) yielded polymers with low dispersity (1.09≤Đ≤1.29) and molecular weights (MWs) close to theoretical values in the presence of pyruvate. Without added pyruvates, lower MWs were observed due to generation of new chains by H2O2 formed by reaction of O2 with GOx. Successful chain extension of POEOMA500 macroinitiator with OEOMA300 (Đ≤1.3) and Bovine Serum Albumin bioconjugates (Đ≤1.22) confirmed a well‐controlled polymerization. The reactions in the open air in larger scale (25 mL) were also successful.
Oxygen‐tolerant: Well‐controlled aqueous atom‐transfer radical polymerization (ATRP) conducted in the open air was made possible by continuous conversion of oxygen to carbon dioxide catalyzed by glucose oxidase (GOx), in the presence of glucose and sodium pyruvate as sequential sacrificial substrates. Without pyruvate lower molecular weights were observed due to the generation of new chains by H2O2 formed by reaction of O2 with GOx.
PEGylation is a well-established and clinically proven half-life extension strategy for protein delivery. Protein modification with amine-reactive poly(ethylene glycol) (PEG) generates heterogeneous ...and complex bioconjugate mixtures, often composed of several PEG positional isomers with varied therapeutic efficacy. Laborious and costly experiments for reaction optimization and purification are needed to generate a therapeutically useful PEG conjugate. Kinetic models which accurately predict the outcome of so-called “random” PEGylation reactions provide an opportunity to bypass extensive wet lab experimentation and streamline the bioconjugation process. In this study, we propose a protein tertiary structure-dependent reactivity model that describes the rate of protein-amine PEGylation and introduces “PEG chain coverage” as a tangible metric to assess the shielding effect of PEG chains. This structure-dependent reactivity model was implemented into three models (linear, structure-based, and machine-learned) to gain insight into how protein-specific molecular descriptors (exposed surface areas, pK a, and surface charge) impacted amine reactivity at each site. Linear and machine-learned models demonstrated over 75% prediction accuracy with butylcholinesterase. Model validation with Somavert, PEGASYS, and phenylalanine ammonia lyase showed good correlation between predicted and experimentally determined degrees of modification. Our structure-dependent reactivity model was also able to simulate PEGylation progress curves and estimate “PEGmer” distribution with accurate predictions across different proteins, PEG linker chemistry, and PEG molecular weights. Moreover, in-depth analysis of these simulated reaction curves highlighted possible PEG conformational transitions (from dumbbell to brush) on the surface of lysozyme, as a function of PEG molecular weight.
Proteins, nucleic acids, lipid vesicles, and carbohydrates are the major classes of biomacromolecules that function to sustain life. Biology also uses post-translation modification to increase the ...diversity and functionality of these materials, which has inspired attaching various other types of polymers to biomacromolecules. These polymers can be naturally (carbohydrates and biomimetic polymers) or synthetically derived and have unique properties with tunable architectures. Polymers are either grafted-to or grown-from the biomacromolecule’s surface, and characteristics including polymer molar mass, grafting density, and degree of branching can be controlled by changing reaction stoichiometries. The resultant conjugated products display a chimerism of properties such as polymer-induced enhancement in stability with maintained bioactivity, and while polymers are most often conjugated to proteins, they are starting to be attached to nucleic acids and lipid membranes (cells) as well. The fundamental studies with protein–polymer conjugates have improved our synthetic approaches, characterization techniques, and understanding of structure–function relationships that will lay the groundwork for creating new conjugated biomacromolecular products which could lead to breakthroughs in genetic and tissue engineering.
Bacteroidetes are a phylum of Gram-negative bacteria abundant in mammalian-associated polymicrobial communities, where they impact digestion, immunity, and resistance to infection. Despite the ...extensive competition at high cell density that occurs in these settings, cell contact-dependent mechanisms of interbacterial antagonism, such as the type VI secretion system (T6SS), have not been defined in this group of organisms. Herein we report the bioinformatic and functional characterization of a T6SS-like pathway in diverse Bacteroidetes. Using prominent human gut commensal and soil-associated species, we demonstrate that these systems localize dynamically within the cell, export antibacterial proteins, and target competitor bacteria. The Bacteroidetes system is a distinct pathway with marked differences in gene content and high evolutionary divergence from the canonical T6S pathway. Our findings offer a potential molecular explanation for the abundance of Bacteroidetes in polymicrobial environments, the observed stability of Bacteroidetes in healthy humans, and the barrier presented by the microbiota against pathogens.
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•Bacterial T6SS divides into three phylogenetically distinct subtypes (T6SSi–iii)•T6SSiii is restricted to Bacteroidetes and is composed of unique components•T6SSiii targets toxic effectors to competing Proteobacteria and other Bacteroidetes•Bacteroides fragilis T6SSiii targets B. thetaiotaomicron and is expressed in vivo
While many Bacteroidetes occupy densely populated niches, including the human gut, cell contact-dependent antagonism of competing bacteria has not been described in this phylum. Russell et al. describe three type VI secretion system subgroups and demonstrate that a subgroup encoded by Bacteroidetes can mediate competition between prominent commensals.
Abstract Rational controlled synthesis of poly(quaternary ammonium) compounds has been used to prepare antimicrobial polymer brushes on inorganic surfaces. The systematic variation of several ...structural parameters of the polymeric brushes allowed us to elicit the minimum surface requirements and a probable mechanism of action for Escherichia coli cell kill. Polymeric brushes were prepared by surface-initiated atom transfer radical polymerization of 2-(dimethylamino)ethyl methacrylate (DMAEMA), a method that allows the molecular weight of the polymer chains to be precisely controlled as they grow from the target surface. The tertiary amino groups of the polyDMAEMA were then quaternized with alkyl bromides to provide a surface with antimicrobial activity. Dry layer thickness of the polymer brushes was controlled by polymerization time and/or initiator density on the surface. This tunability of surface structure allows the antimicrobial polymer brushes to be tailored rationally. A combinatorial screening tool was developed to elucidate the role of chain length and chain density on cell kill in a single experiment. The results indicate that surface charge density, is a critical element in designing a surface for maximum kill efficiency. The most biocidal surfaces had charge densities of greater than 1–5×1015 accessible quaternary amine units/cm2 . The relevance of this finding to the mechanism of action is discussed.
Atom transfer radical polymerization (ATRP) can be carried out in a flask completely open to air using a biocatalytic system composed of glucose oxidase (GOx) and horseradish peroxidase (HRP) with an ...active copper catalyst complex. Nanomolar concentrations of the enzymes and ppm amounts of Cu provided excellent control over the polymerization of oligo(ethylene oxide) methyl ether methacrylate (OEOMA500), generating polymers with high molecular weight (Mn>70 000) and low dispersities (1.13≤Đ≤1.27) in less than an hour. The continuous oxygen supply was necessary for the generation of radicals and polymer chain growth as demonstrated by temporal control and by inducing hypoxic conditions. In addition, the enzymatic cascade polymerization triggered by oxygen was used for a protein and DNA functionalized with initiators to form protein‐b‐POEOMA and DNA‐b‐POEOMA bioconjugates, respectively.
Oxygen keeps polymerization alive: An oxygen‐dependent atom transfer radical polymerization was accomplished through the biocatalytic mediation of radicals by a glucose oxidase/horseradish peroxidase system “orchestrated” by a Cu/tris(2‐pyridylmethyl)amine (TPMA) catalyst (see picture; ACAC=acetylacetonate). This system provides polymers with high molecular weights and low dispersities, and is compatible with biologically relevant environments.
In this study, we report on multimodal temperature-responsive chymotrypsin-poly(sulfobetaine methacrylamide)-block-poly(N-isopropylacrylamide) (CT-pSBAm-block-pNIPAm) protein–polymer conjugates. ...Using polymer-based protein engineering (PBPE) with aqueous atom transfer radical polymerization (ATRP), we synthesized three different molecular weight CT-pSBAm-block-pNIPAm bioconjugates that responded structurally to both low and high temperature. In the block copolymer grown from the surface of the enzyme, upper critical solution temperature (UCST) phase transition was dependent on the chain length of the polymers in the conjugates, whereas lower critical solution temperature (LCST) phase transition was independent of molecular weight. Each CT-pSBAm-block-pNIPAm conjugate showed temperature dependent changes in substrate affinity and productivity when assayed from 0 to 40 °C. In addition, these conjugates showed higher stability to harsh conditions, including temperature, low pH, and protease degradation. Indeed, the PBPE-modified enzyme was active for over 8 h in the presence of a stomach protease at pH 1.0. Using PBPE, we created a dual zone shell surrounding each molecule of enzyme. The thickness of each zone of the shell was engineered to be separately responsive to temperature.
Abstract Polymer-based protein engineering (PBPE) offers an attractive method to predictably modify and enhance enzyme structure and function. Using polymers that respond to stimuli such as ...temperature and pH, enzyme activity and stability can be predictably modified without a dependence on molecular biology. Herein, we demonstrate that temperature responsive enzyme-polymer conjugates show increased stability while retaining bioactivity and substrate affinity. The bioconjugates were synthesized using a “grafting from” approach, where polymers were grown from a novel water-soluble initiator on the surface of a protein using atom transfer radical polymerization. Prior to polymer synthesis, the polymerization initiating molecule was covalently attached to surface accessible primary amines (lysine, N-terminal) of chymotrypsin, forming a macroinitiator. Poly( N -isopropylacrylamide) and poly N , N’ -dimethyl(methacryloylethyl) ammonium propane sulfonate were grown separately from the initiator modified chymotrypsin. Both polymers were selected because of their temperature-dependent conformations. We observed that the enzyme-polymer conjugates retained temperature-dependent changes in conformation while still maintaining enzyme function. The conjugates exhibited dramatic increases in enzyme stability over a wide range of temperatures. We can now predictably manipulate enzyme kinetics and stability using polymer-based protein engineering without the need for molecular biology dependent mutagenesis.
The last decade has seen an exponential expansion of interest in conjugating multiple enzymes of cascades in close proximity to each other, with the overarching goal being to accelerate the overall ...reaction rate. However, some evidence has emerged that there is no effect of proximity channeling on the reaction velocity of the popular GOx-HRP cascade, particularly in the presence of a competing enzyme (catalase). Herein, we rationalize these experimental results quantitatively. We show that, in general, proximity channeling can enhance reaction velocity in the presence of competing enzymes, but in steady state a significant enhancement can only be achieved for diffusion-limited reactions or at high concentrations of competing enzymes. We provide simple equations to estimate the effect of channeling quantitatively and demonstrate that proximity can have a more pronounced effect under crowding conditions in vivo, particularly that crowding can enhance the overall rates of channeled cascade reactions.
Antigenic and genetic analysis of the hemagglutinin of ~13,000 human influenza A (H3N2) viruses from six continents during 2002-2007 revealed that there was continuous circulation in east and ...Southeast Asia (E-SE Asia) via a region-wide network of temporally overlapping epidemics and that epidemics in the temperate regions were seeded from this network each year. Seed strains generally first reached Oceania, North America, and Europe, and later South America. This evidence suggests that once A (H3N2) viruses leave E-SE Asia, they are unlikely to contribute to long-term viral evolution. If the trends observed during this period are an accurate representation of overall patterns of spread, then the antigenic characteristics of A (H3N2) viruses outside E-SE Asia may be forecast each year based on surveillance within E-SE Asia, with consequent improvements to vaccine strain selection.
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