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Airborne and surface Pb concentrations were measured inside a glovebox chamber with controlled inflow rate at 1.0 L/min of ultrapure air over a 4-hour period while an operator completed 1,680 solde...

Dual-associative protein di- and triblock copolymers composed of sticker-decorated midblocks and micelle-forming elastin-like polypeptide (ELP) endblocks form shear-thinning, thermoresponsively reinforceable hydrogels that are potentially useful as injectable materials for a variety of applications. Here, the combination of rheological and in situ scattering measurements under shear on these dual-associative gels is employed in order to better understand how block architecture plays a role in controlling microscopic structural rearrangement and the resulting macroscopic mechanical responses. These gels, which form a disordered sphere phase due to endblock aggregation under quiescent conditions with the midblock domains physically crosslinked by protein associations, exhibit both viscoelastic and thixotropic signatures with relative magnitudes dependent upon gel concentration and block architecture. In situ SAXS measurements during flow indicate that these thixotropic responses correspond to the development of ordered domains following start-up of shear. For both architectures, the rate of alignment increases with increasing concentration. However, the rate of domain formation when increasing the temperature from 35 to 50 °C depends on the interplay between thermoresponsive toughening of the endblocks and softening of the coiled-coil domains such that rate of rearrangement decreases in the triblock while it increases in the diblock. Following a step-down in shear flow, structural rearrangement within the samples results in a thixotropic stress response. Upon cessation of flow, gel recovery is characterized by a concentration-dependent restoration of the micellar network over time, with two timescales observed that correspond to two different length scales of network relaxation.

Nucleoporin-like polypeptide (NLP) hydrogels are developed by mimicking nucleoporins, proteins that form gel filters regulating transport into the nucleus. Using protein polymers of a minimal consensus repeat, the NLPs selectively enhance transport of cargo–carrier complexes similar to the natural nuclear pore system. The engineered protein gels additionally have tunable mechanical and transport properties and can be biosynthesized at high yield, making them promising materials for advanced separation technologies., United States. Defense Threat Reduction Agency (Grant HDTRA1-13-1-0038), National Institutes of Health (U.S.) (Grant 5-T32-GM008834), National Institute for Biomedical Imaging and Bioengineering (U.S.) (Grant P41EB015871-28), MIT Skoltech Initiative

Polymers exhibiting lower critical solution behavior in water have found broad use as thermoresponsive moieties in soft materials, particularly in biomedical applications for triggered actuation, gelation, accumulation, or release. In this work, changing the thermoresponsive block in a self-assembling hydrogel is shown to be a useful approach to control the viscoelastic behavior and mechanical reinforcement of the gel above its transition temperature. Triblock copolymers were prepared with artificial associative protein midblocks from either site-specific bioconjugation of narrowly disperse poly(N-isopropylacrylamide) (PNIPAM) or as biosynthetic genetic fusions to monodisperse elastin-like polypeptide (ELP) sequences. Both synthetic approaches yield responsively reinforceable hydrogels that can be stiffened by up to an order of magnitude to approximately 105 Pa at 30% (w/w). However, end block chemical composition and linear block copolymer architecture could be manipulated to yield high-temperature plate...

Perfectly defined, monodisperse fusion protein block copolymers of a thermoresponsive coil-like protein, ELP, and a globular protein, mCherry, are demonstrated to act as fully biosynthetic analogues to protein-polymer conjugates that can self-assemble into biofunctional nanostructures such as hexagonal and lamellar phases in concentrated solutions. The phase behavior of two mCherry-ELP fusions, E10-mCherry-E10 and E20-mCherry, is investigated to compare linear and bola fusion self-assembly both in diluted and concentrated aqueous solution. In dilute solution, the molecular topology impacts the stability of micelles formed above the thermal transition temperature of the ELP block, with the diblock forming micelles and the bola forming unstable aggregates. Despite the chemical similarity of the two protein blocks, the materials order into block copolymer-like nanostructures across a wide range of concentrations at 30 wt% and above, with the bola fusion having a lower order-disorder transition concentration than the diblock fusion. The topology of the molecule has a large impact on the type of nanostructure formed, with the two fusions forming phases in the opposite order as a function of temperature and concentration. This new system provides a rich landscape to explore the capabilities of fusion architecture to control supramolecular assemblies for bioactive materials.

Formulation of tissue engineering or regenerative scaffolds from simple bioactive polymers with tunable structure and mechanics is crucial for the regeneration of complex tissues, and hydrogels from recombinant proteins, such as elastin-like polypeptides (ELPs), are promising platforms to support these applications. The arrested phase separation of ELPs has been shown to yield remarkably stiff, biocontinuous, nanostructured networks, but these gels are limited in applications by their relatively brittle nature. Here, a gel-forming ELP is chain-extended by telechelic oxidative coupling, forming extensible, tough hydrogels. Small angle scattering indicates that the chain-extended polypeptides form a fractal network of nanoscale aggregates over a broad concentration range, accessing moduli ranging from 5 kPa to over 1 MPa over a concentration range of 5-30 wt %. These networks exhibited excellent erosion resistance and allowed for the diffusion and release of encapsulated particles consistent with a bicontinuous, porous structure with a broad distribution of pore sizes. Biofunctionalized, toughened networks were found to maintain the viability of human mesenchymal stem cells (hMSCs) in 2D, demonstrating signs of osteogenesis even in cell media without osteogenic molecules. Furthermore, chondrocytes could be readily mixed into these gels via thermoresponsive assembly and remained viable in extended culture. These studies demonstrate the ability to engineer ELP-based arrested physical networks on the molecular level to form reinforced, cytocompatible hydrogel matrices, supporting the promise of these new materials as candidates for the engineering and regeneration of stiff tissues.

The preparation of new responsive hydrogels is crucial for the development of soft materials for various applications, including additive manufacturing and biomedical implants. Here, we report the discovery of a new mechanism for forming physical hydrogels by the arrested phase separation of a subclass of responsively hydrophobic elastin-like polypeptides (ELPs). When moderately concentrated solutions of ELPs with the pentapeptide repeat (XPAVG)n (where X is either 20% or 60% valine with the remainder isoleucine) are warmed above their inverse transition temperature, phase separation becomes arrested, and hydrogels can be formed with shear moduli on the order of 0.1-1 MPa at 20 wt % in water. The longest stress relaxation times are well beyond 10(3) s. This result is surprising because ELPs are classically known for thermoresponsive coacervation that leads to macrophase separation, and solids are typically formed in the bulk or by supplemental cross-linking strategies. This new mechanism can form gels with remarkable mechanical behavior based on simple macromolecules that can be easily engineered. Small angle scattering experiments indicate that phase separation arrests to form a network of nanoscale domains, exhibiting rheological and structural features consistent with an arrested spinodal decomposition mechanism. Gel nanostructure can be modeled as a disordered bicontinuous network with interdomain, intradomain, and curvature length scales that can be controlled by sequence design and assembly conditions. These studies introduce a new class of reversible, responsive materials based on a classic artificial biopolymer that is a versatile platform to address critical challenges in industrial and medical applications.

Molecular defects critically impact the properties of materials. Here we introduce a paradigm called "isotopic labeling disassembly spectrometry" (ILDaS) that facilitates unprecedented precise experimental correlations between elastically inactive network defects (dangling chains and primary loops) and network formation kinetics and precursor structure. ILDaS is inspired by classical crossover experiments, which are often used to interrogate whether a reaction mechanism proceeds via an inter- or intramolecular pathway. We show that if networks are designed from labeled bifunctional monomers that transfer their labels to multifunctional junctions upon network formation, then the extent of junction labeling correlates directly with the number of dangling chains and cyclic imperfections within the network. We demonstrate two complementary ILDaS approaches that enable defect measurements with short analysis times, low cost, and synthetic versatility applicable to a broad range of network materials including polydisperse polymer precursors. The results will spur new experimental and theoretical investigations into the interplay between polymer network structure and properties.

Engineering artificial protein hydrogels for medical applications requires precise control over their mechanical properties, including stiffness, toughness, extensibility, and stability in the physiological environment. Here we demonstrate topological entanglement as an effective strategy to robustly increase the mechanical tunability of a transient hydrogel network based on coiled-coil interactions. Chain extension and entanglement are achieved by coupling the cysteine residues near the N- and C-termini, and the resulting chain distribution is found to agree with the Jacobson–Stockmayer theory. By exploiting the reversible nature of the disulfide bonds, the entanglement effect can be switched on and off by redox stimuli. With the presence of entanglements, hydrogels exhibit a 7.2-fold enhanced creep resistance and a suppressed erosion rate by a factor of 5.8, making the gels more mechanically stable in a physiologically relevant open system. While hardly affecting material stiffness (only resulting in a 1.5-fold increase in the plateau modulus), the entanglements remarkably lead to hydrogels with a toughness of 65 000 J m^(–3) and extensibility to approximately 3000% engineering strain, which enables the preparation of tough yet soft tissue simulants. This improvement in mechanical properties resembles that from double-network hydrogels but is achieved with the use of a single associating network and topological entanglement. Therefore, redox-triggered chain entanglement offers an effective approach for constructing mechanically enhanced and responsive injectable hydrogels.

Shear thinning hydrogels are promising materials that exhibit rapid self-healing following the cessation of shear, making them attractive for a variety of applications including injectable biomaterials. In this work, self-assembly is demonstrated as a strategy to introduce a reinforcing network within shear thinning artificially engineered protein gels, enabling a responsive transition from an injectable state at low temperatures with a low yield stress to a stiffened state at physiological temperatures with resistance to shear thinning, higher toughness, and reduced erosion rates and creep compliance. Protein-polymer triblock copolymers capable of the responsive self-assembly of two orthogonal networks have been synthesized by conjugating poly(N-isopropylacrylamide) to the N- and C- termini of a protein midblock decorated with coiled-coil self-associating domains. Midblock association forms a shear-thinning network, while endblock aggregation at elevated temperatures introduces a second, independent physical network into the protein hydrogel. These new, reversible crosslinks introduce extremely long relaxation times and lead to a five-fold increase in the elastic modulus, significantly larger than is expected from transient network theory. Thermoresponsive reinforcement reduces the high temperature creep compliance by over four orders of magnitude, decreases the erosion rate by at least a factor of five, and increases the yield stress by up to a factor of seven. The reinforced hydrogels also exhibit enhanced resistance to plastic deformation and failure in uniaxial compression. Combined with the demonstrated potential of shear thinning artificial protein hydrogels for various uses, including the minimally-invasive implantation of bioactive scaffolds, this reinforcement mechanism broadens the range of applications that can be addressed with shear-thinning physical gels.

Self-assembly of three-dimensional solid-state nanostructures containing approximately 33% by weight globular protein is demonstrated using a globular protein-polymer diblock copolymer, providing a route to direct nanopatterning of proteins for use in bioelectronic and biocatalytic materials. A mutant red fluorescent protein, mCherryS131C, was prepared by incorporation of a unique cysteine residue and site-specifically conjugated to end-functionalized poly(N-isopropylacrylamide) through thiol-maleimide coupling to form a well-defined model protein-polymer block copolymer. The block copolymer was self-assembled into bulk nanostructures by solvent evaporation from concentrated solutions. Small-angle X-ray scattering and transmission electron microscopy illustrated the formation of highly disordered lamellae or hexagonally perforated lamellae depending upon the selectivity of the solvent during evaporation. Solvent annealing of bulk samples resulted in a transition towards lamellar nanostructures with mCherry packed in a bilayer configuration and a large improvement in long range ordering. Wide-angle X-ray scattering indicated that mCherry did not crystallize within the block copolymer nanodomains and that the β-sheet spacing was not affected by self-assembly. Circular dichroism showed no change in protein secondary structure after self-assembly, while UV-vis spectroscopy indicated approximately 35% of the chromophore remained optically active.

A strategy for responsively toughening an injectable protein hydrogel has been implemented by incorporating an associative protein as the midblock in triblock copolymers with thermoresponsive poly(N-isopropylacrylamide) (PNIPAM) endblocks, producing materials with a low yield stress necessary for injectability and durability required for load-bearing applications post-injection. Responsive reinforcement triggered by PNIPAM association leads to significant increases in the gel’s elastic modulus as well as its resistance to creep. The performance of these materials is a strong function of molecular design, with certain formulations reaching elastic moduli of up to 130 kPa, effectively reinforced by a factor of 14 over their low temperature moduli, and having stress relaxation times increased by up to a factor of 50. The nanostructural origins of these thermoresponsive enhancements were explored, demonstrating that large micellar cores, high PNIPAM volume fractions, and high densities of associating groups in the protein corona lead to the greatest reinforcement of the gel’s elastic modulus. Gels with the largest micelles and the highest packing fractions also had the longest relaxation times in the reinforced state. These combined structure and mechanics studies reveal that control of both the micellar and protein networks is critical for making high performance gels relevant for biomedical applications.

One selection pressure shaping sequence evolution is the requirement that a protein fold with sufficient stability to perform its biological functions. We present a conceptual framework that explains how this requirement causes the probability that a particular amino acid mutation is fixed during evolution to depend on its effect on protein stability. We mathematically formalize this framework to develop a Bayesian approach for inferring the stability effects of individual mutations from homologous protein sequences of known phylogeny. This approach is able to predict published experimentally measured mutational stability effects (ΔΔG values) with an accuracy that exceeds both a state-of-the-art physicochemical modeling program and the sequence-based consensus approach. As a further test, we use our phylogenetic inference approach to predict stabilizing mutations to influenza hemagglutinin. We introduce these mutations into a temperature-sensitive influenza virus with a defect in its hemagglutinin gene and experimentally demonstrate that some of the mutations allow the virus to grow at higher temperatures. Our work therefore describes a powerful new approach for predicting stabilizing mutations that can be successfully applied even to large, complex proteins such as hemagglutinin. This approach also makes a mathematical link between phylogenetics and experimentally measurable protein properties, potentially paving the way for more accurate analyses of molecular evolution., Author Summary Mutating a protein frequently causes a change in its stability. As scientists, we often care about these changes because we would like to engineer a protein's stability or understand how its stability is impacted by a naturally occurring mutation. Evolution also cares about mutational stability changes, because a basic evolutionary requirement is that proteins remain sufficiently stable to perform their biological functions. Our work is based on the idea that it should be possible to use the fact that evolution selects for stability to infer from related proteins the effects of specific mutations. We show that we can indeed use protein evolutionary histories to computationally predict previously measured mutational stability changes more accurately than methods based on either of the two main existing strategies. We then test whether we can predict mutations that increase the stability of hemagglutinin, an influenza protein whose rapid evolution is partly responsible for the ability of this virus to cause yearly epidemics. We experimentally create viruses carrying predicted stabilizing mutations and find that several do in fact improve the virus's ability to grow at higher temperatures. Our computational approach may therefore be of use in understanding the evolution of this medically important virus.