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As proteins are abundant polymers in biomass sources such as agricultural feedstocks and byproducts, leveraging them to develop alternatives to synthetic polymers is of great interest. However, the mechanical performance of protein materials is not suitable for most target applications. Constructing copolymers with proteins as hard domains and rubbery polymers as soft domains has been shown to be a promising strategy for improving mechanical properties. Herein, it is demonstrated that toughening and strengthening of protein copolymers can be advanced further by thermal treatment, leading to mechanical enhancements that generalize across a variety of different protein feedstocks, including whey, serum, soy, and pea proteins. The thermal treatment induces a rearrangement of protein structure, leading to the formation of intermolecular β-sheets. The ordered intermolecular structures in the hard domains of thermosets greatly improve their mechanical properties, providing simultaneous increases in strength, toughness, and modulus, with little sacrifice in fracture strain. Analogous to crystalline structures, the formation of intermolecular β-sheet structures also leads to reduced hygroscopicity. This is a valuable contribution, as practical applications of natural polymer-based plastics are frequently hindered by the materials' humidity sensitivity. Therefore, this work demonstrates a simple yet versatile strategy to improve the materials' performance from a wide range of protein feedstocks, as well as signifies the implications of protein structural assembly in materials design.

Natural selective filtering systems (e.g., the extracellular matrix, nuclear pores, and mucus) separate molecules selectively and efficiently, and the detailed understanding of transport mechanisms exploited in these systems provides important bioinspired design principles for selective filters. In particular, nucleoporins consist of consensus repeat sequences that are readily utilized for engineering repeat proteins. Here, the consensus repeat sequence of Nsp1, a yeast nucleoporin, is polymerized to form a nucleoporin-like protein (NLP) and mutated to understand the effect of sequence on selective transport. The hydrophilic spacers of the NLPs were redesigned considering net charge, charge distribution, and polarity. Mutations were made near to and far from the FSFG interacting domain to explore the role of highly conserved residues as a function of spatial proximity. A nuclear transport receptor-cargo complex, nuclear transport factor 2-green fluorescent protein (NTF2-GFP), was used as a model for changes in transport. For mutations of the charged spacer, some mutations of highly conserved charged residues were possible without knocking out selective transport of the NTF2, but the formation of regions of clustered negative charge has an unfavorable effect on nuclear transporter permeation. Thus, positive net charge and alternating positive and negative charge within the hydrophilic spacer are advantageous for recognition and selective transport. In the polarity panel, mutations that increased the interaction between NTF2-GFP and the gel led to decreased permeation of the NTF2-GFP due to blocking of the interface and inability of the NTF2-GFP to transport into the gel. Therefore, these results provide a strategy for tuning selective permeability of biomolecules using the artificially designed consensus repeat-based hydrogels.

Protein-polymer bioconjugate self-assembly has attracted a great deal of attention as a method to fabricate protein nanomaterials in solution and the solid state. To identify protein properties that affect phase behavior in protein-polymer block copolymers, a library of 15 unique protein-b-poly(N-isopropylacrylamide) (PNIPAM) copolymers comprising 11 different proteins was compiled and analyzed. Many attributes of phase behavior are found to be similar among all studied bioconjugates regardless of protein properties, such as formation of micellar phases at high temperature and low concentration, lamellar ordering with increasing temperature, and disordering at high concentration, but several key protein-dependent trends are also observed. In particular, hexagonal phases are only observed for proteins within the molar mass range 20-36 kDa, where ordering quality is also significantly enhanced. While ordering is generally found to improve with increasing molecular weight outside of this range, most large bioconjugates exhibited weaker than predicted assembly, which is attributed to chain entanglement with increasing polymer molecular weight. Additionally, order-disorder transition boundaries are found to be largely uncorrelated to protein size and quality of ordering. However, the primary finding is that bioconjugate ordering can be accurately predicted using only protein molecular weight and percentage of residues contained within β sheets. This model provides a basis for designing protein-PNIPAM bioconjugates that exhibit well-defined self-assembly and a modeling framework that can generalize to other bioconjugate chemistries., United States. Department of Energy. Office of Basic Energy Sciences (Award DE-SC0007106)

Charge anisotropy or the presence of charge patches at protein surfaces has long been thought to shift the coacervation curves of proteins and has been used to explain the ability of some proteins to coacervate on the "wrong side" of their isoelectric point. This work makes use of a panel of engineered superfolder green fluorescent protein mutants with varying surface charge distributions but equivalent net charge and a suite of strong and weak polyelectrolytes to explore this concept. A patchiness parameter, which assessed the charge correlation between points on the surface of the protein, was used to quantify the patchiness of the designed mutants. Complexation between the polyelectrolytes and proteins showed that the mutant with the largest patchiness parameter was the most likely to form complexes, while the smallest was the least likely to do so. The patchiness parameter was found to correlate well with the phase behavior of the protein-polymer mixtures, where both macrophase separation and the formation of soluble aggregates were promoted by increasing the patchiness depending on the polyelectrolyte with which the protein was mixed. Increasing total charge and increasing strength of the polyelectrolyte promote interactions for oppositely charged polyelectrolytes, while charge regulation is also key to interactions for similarly charged polyelectrolytes, which must interact selectively with oppositely charged patches.

Biological systems routinely regulate biomolecular transport with remarkable specificity, low energy input, and simple mechanisms. Here, the biophysical mechanisms of nuclear transport inspire the development of gels for recognition and selective permeation (GRASP). GRASP presents a new paradigm for specific transport and selective permeability, in which binding interactions between a biomolecule and a hydrogel lead to faster penetration of the gel. A molecular transport theory identifies key principles for selective transport: entropic repulsion of noninteracting molecules and affinity-mediated diffusion of multireceptor biomolecules through a walking mechanism. The ability of interacting molecules to walk through hydrogels enables enhanced permeability in polymer networks. To realize this theoretical prediction in a novel material, GRASP is engineered from a poly(ethylene glycol) network (entropic barrier) containing antibody-binding oligopeptides (affinity domains). GRASP is synthesized using simultaneous bioconjugation and polycondensation reactions. The elastic modulus, characteristic pore size, biomolecular diffusivity, and selective permeability are measured in the resulting materials, which are applied to regulate the transport of equally sized molecules by preferentially transporting a monoclonal antibody from a polyclonal mixture. Overall, this work presents rationally designed, nucleopore-inspired hydrogels that are capable of controlling biomolecular transport.

The self-assembly of protein-polymer conjugates incorporating oligomers of a small, engineered high-affinity binding protein, rcSso7d.SA, is studied to determine the effect of protein oligomerization on nanoscale ordering. Oligomerization enables a systematic increase in the protein molar mass without changing its overall folded structure, leading to a higher driving force for self-assembly into well-ordered structures. Though conjugates of monomeric rcSso7d.SA are found to only exist in disordered states, oligomers of this protein linked to a poly( N-isopropylacrylamide) (PNIPAM) block self-assemble into lamellar nanostructures. Conjugates of trimeric and tetrameric rcSso7d.SA are observed to produce the strongest ordering in concentrated solution, displaying birefringent lamellae at concentrations as low as 40 wt %. In highly concentrated solution, the oligomeric rcSso7d.SA-PNIPAM block copolymers exhibit ordering and domain spacing trends atypical from that of most block copolymers. Fluorescent binding assays indicate that oligomerized protein blocks retain binding functionality and exhibit limits of detection up to three times lower than that of surface-immobilized protein sensors. Therefore, oligomerization of the protein block in these block copolymers serves as an effective method to improve both nanoscale ordering and biosensing capabilities.

Elastin-like polypeptides (ELPs) are one of the most widely-studied classes of protein material because of their lower critical solution temperature (LCST)-like thermoresponsive behavior in aqueous solutions. Here, it is shown that ELPs also exhibit cononsolvency effects, similar to many other water-soluble polymers. The effect of solvent composition on the dilute solution phase behavior of an elastin-like polypeptide is studied here in water/alcohol blends that contain 0-40 vol % methanol, ethanol, isopropanol, or 1-propanol. In all systems studied, the ELP exhibits cononsolvency behavior at low alcohol content, as indicated by a decrease in the transition temperature of the ELP. When the alcohol added is ethanol, isopropanol, or 1-propanol, the decrease in transition temperature is followed by a region of complete ELP insolubility, and, finally, the emergence of upper-critical solution transition (UCST)-like behavior. The ELP is completely soluble at all temperatures measured at alcohol contents above 40 vol %. The effect of sodium chloride on this ELP cononsolvency in water/ethanol blends was also studied. Unlike the previously studied polymer poly( N-isoropylacrylamide) (PNIPAM), ELP exhibits nonmonotonic changes in transition temperature with the addition of sodium chloride at ethanol contents that produce UCST-like transitions of the ELP. This discovery of ELP cononsolvency in water/alcohol systems introduces a new handle with which the solubility of ELPs can be tuned.

Mutation of a superfolder green fluorescent protein (GFP) was used to design GFP variants with formal net charges of 0, -8, and -21, providing a set of three proteins in which the total charge is varied to tune protein-protein interactions while controlling for the protein size and tertiary structure. After conjugating poly(N-isopropylacrylamide) (PNIPAM) to each of these three GFP variants, the concentrated solution phase behavior of these three block copolymers is studied using a combination of small-angle X-ray scattering (SAXS), depolarized light scattering (DPLS), and turbidimetry to characterize their morphologies. The electrostatic repulsion between supercharged GFP suppresses ordering, increasing the order-disorder transition concentration (CODT) and decreasing the quality of the ordered nanostructures as measured by the full width at half-maximum of the primary scattering peak. By contrast, the charge distribution of the neutrally charged GFP results in its largest dipole moment, calculated about the protein's center of mass, among the three GFP variants and a self-complementary Janus-like electrostatic surface potential that enhances nanostructure formation. The different electrostatic properties result in different protein-protein interactions that affect the high temperature morphologies, including the formation of macrophase separated or homogeneous micellar phases and the smaller hexagonal ordering window of the supercharged GFP. Small improvements in the quality of the ordered nanostructures of GFP(-21)-PNIPAM can be achieved through protein-divalent cation interactions. Therefore, varying protein charge and electrostatics is demonstrated as a method of tuning the magnitude and directionality of protein-protein interactions to control self-assembly.

Fusion proteins provide a facile route for the purification and self-assembly of biofunctional protein block copolymers into complex nanostructures; however, the use of biochemical synthesis techniques introduces unexplored variables into the design of the structures. Using model fusion constructs of the red fluorescent protein mCherry and the coil-like protein elastin-like polypeptide (ELP), it is shown that the molar mass and hydrophobicity of the ELP sequence have a large effect on the propensity of a fusion to form well-ordered nanostructures, even when the ELP is in the low temperature, highly solvated state. In contrast, the presence of a 6xHis purification tag has little effect on self-assembly, and the order of blocks in the construct (N-terminal vs C-terminal) only has a significant effect on the nanostructure when the conjugates are heated above the transition temperature of the ELP block. These results indicate that for a sufficiently hydrophobic and high molar mass ELP block, there is a great deal of design latitude in the construction of fusion protein block copolymers for self-assembling nanomaterials.

Self-assembly of protein-polymer bioconjugates presents an elegant strategy for controlling nanostructure and orientation of globular proteins in functional materials. Recent work has shown that genetic fusion of globular protein mCherry to an elastin-like polypeptide (ELP) yields similar self-assembly behavior to these protein-polymer bioconjugates. In the context of studying protein-polymer bioconjugate self-assembly, the mutability of the ELP sequence allows several different properties of the ELP block to be tuned orthogonally while maintaining consistent polypeptide backbone chemistry. This work uses this ELP sequence tunability in combination with the precise control offered by genetic engineering of an amino acid sequence to generate a library of four novel ELP sequences that are used to study the combined effect of charge and hydrophobicity on ELP-mCherry fusion protein self-assembly. Concentrated solution self-assembly is studied by small-angle X-ray scattering (SAXS) and depolarized light scattering (DPLS). These experiments show that fusions containing a negatively charged ELP block do not assemble at all, and fusions with a charge balanced ELP block exhibit a weak propensity for assembly. By comparison, the fusion containing an uncharged ELP block starts to order at 40 wt % in solution and at all concentrations measured has sharper, more intense SAXS peaks than other fusion proteins. These experiments show that charge character of the ELP block is a stronger predictor of self-assembly behavior than the hydrophobicity of the ELP block. Dilute solution small-angle neutron scattering (SANS) on the ELPs alone suggests that all ELPs used in this study (including the uncharged ELP) adopt dilute solution conformations similar to those of traditional polymers, including polyampholytes and polyelectrolytes. Finally, dynamic light scattering studies on ELP-mCherry blends shows that there is no significant complexation between the charged ELPs and mCherry. Therefore, it is proposed that the superior self-assembly of fusion proteins containing uncharged ELP block is due to effective repulsions between charged and uncharged blocks due to local charge correlation effects and, in the case of anionic ELPs, repulsion between like charges within the ELP block.

Blending the small molecule osmolytes glycerol and trehalose with the model globular protein-polymer block copolymer mCherry-b-poly(N-isopropyl acrylamide) (mCherry-b-PNIPAM) is demonstrated to improve protein functionality in self-assembled nanostructures. The incorporation of either additive into block copolymers results in functionality retention in the solid state of 80 and 100% for PNIPAM volume fractions of 40 and 55%, respectively. This represents a large improvement over the 50-60% functionality observed in the absence of any additive. Furthermore, glycerol decreases the thermal stability of block copolymer films by 15-20 °C, while trehalose results in an improvement in the thermal stability by 15-20 °C. These results suggest that hydrogen bond replacement is responsible for the retention of protein function but suppression or enhancement of thermal motion based on the glass transition of the osmolyte primarily determines thermal stability. While both osmolytes are observed to have a disordering effect on the nanostructure morphology with increasing concentration, this effect is less pronounced in materials with a larger polymer volume fraction. Glycerol preferentially localizes in the protein domains and swells the nanostructures, inducing disordering or a change in morphology depending on the PNIPAM coil fraction. In contrast, trehalose is observed to macrophase separate from the block copolymer, which results in nanodomains becoming more disordered without changing significantly in size.

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.

Gelation of the left helical N-substituted homopolypeptide poly(l-proline) (PLP) in water was explored, employing rheological and small-angle scattering studies at different temperatures and concentrations in order to investigate the network structure and its mechanical properties. Stiff gels were obtained at 10 wt % or higher at 5 °C, the first time gelation has been observed for homopolypeptides. The secondary structure and helical rigidity of PLP has large structural similarities to gelatin but as gels the two materials show contrasting trends with temperature. With increasing temperature in D2O, the network stiffens, with broad scattering features of similar correlation length for all concentrations and molar masses of PLP. A thermoresponsive transition was also achieved between 5 and 35 °C, with moduli at 35 °C higher than gelatin at 5 °C. The brittle gels could tolerate strains of 1% before yielding with a frequency-independent modulus over the observed range, similar to natural proline-rich proteins, suggesting the potential for thermoresponsive or biomaterial-based applications.

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.

The effects of protein surface potential on the self-assembly of protein-polymer block copolymers are investigated in globular proteins with controlled shape through two approaches: comparison of self-assembly of mCherry-poly(N-isopropylacrylamide) (PNIPAM) bioconjugates with structurally homologous enhanced green fluorescent protein (EGFP)-PNIPAM bioconjugates, and mutants of mCherry with altered electrostatic patchiness. Despite large changes in amino acid sequence, the temperature-concentration phase diagrams of EGFP-PNIPAM and mCherry-PNIPAM conjugates have similar phase transition concentrations. Both materials form identical phases at two different coil fractions below the PNIPAM thermal transition temperature and in the bulk. However, at temperatures above the thermoresponsive transition, mCherry conjugates form hexagonal phases at high concentrations while EGFP conjugates form a disordered micellar phase. At lower concentration, mCherry shows a two-phase region while EGFP forms homogeneous disordered micellar structures, reflecting the effect of changes in micellar stability. Conjugates of four mCherry variants with changes to their electrostatic surface patchiness also showed minimal change in phase behavior, suggesting that surface patchiness has only a small effect on the self-assembly process. Measurements of protein/polymer miscibility, second virial coefficients, and zeta potential show that these coarse-grained interactions are similar between mCherry and EGFP, indicating that coarse-grained interactions largely capture the relevant physics for soluble, monomeric globular protein-polymer conjugate self-assembly.