Engineered Proteins and Materials Utilizing Residue-Specific Noncanonical Amino Acid Incorporation.

The incorporation of noncanonical amino acids into proteins and protein-based materials has significantly expanded the repertoire of available protein structures and chemistries. Through residue-specific incorporation, protein properties can be globally modified, resulting in the creation of novel proteins and materials with diverse and tailored characteristics. In this review, we highlight recent advancements in residue-specific incorporation techniques as well as the applications of the engineered proteins and materials. Specifically, we discuss their utility in bio-orthogonal noncanonical amino acid tagging (BONCAT), fluorescent noncanonical amino acid tagging (FUNCAT), threonine-derived noncanonical amino acid tagging (THRONCAT), cross-linking, fluorination, and enzyme engineering. This review underscores the importance of noncanonical amino acid incorporation as a tool for the development of tailored protein properties to meet diverse research and industrial needs.

Structure-Dependent Water Responsiveness of Protein Block Copolymers.

Biological water-responsive (WR) materials are abundant in nature, and they are used as mechanical actuators for seed dispersal by many plants such as wheat awns and pinecones. WR biomaterials are of interest for applications as high-energy actuators, which can be useful in soft robotics or for capturing energy from natural water evaporation. Recent work on WR silk proteins has shown that ß-sheet nanocrystalline domains with high stiffness correlate with the high WR actuation energy density, but the fundamental mechanisms to drive water responsiveness in proteins remain poorly understood. Here, we design, synthesize, and study protein block copolymers consisting of two α-helical domains derived from cartilage oligomeric matrix protein coiled-coil (C) flanking an elastin-like peptide domain (E), namely, CEC. We use these protein materials to create WR actuators with energy densities that outperform mammalian muscle. To elucidate the effect of structure on WR actuation, CEC was compared to a variant, CECL44A, in which a point mutation disrupts the α-helical structure of the C domain. Surprisingly, CECL44A outperformed CEC, showing higher energy density and less susceptibility to degradation after repeated cycling. We show that CECL44A exhibits a higher degree of intermolecular interactions and is stiffer than CEC at high relative humidity (RH), allowing for less energy dissipation during water responsiveness. These results suggest that strong intermolecular interactions and the resulting, relatively steady protein structure are important for water responsiveness.

Engineered coiled-coil HIF1α protein domain mimic.

The development of targeted anti-cancer therapeutics offers the potential for increased efficacy of drugs and diagnostics. Utilizing modalities agnostic to tumor type, such as the hypoxic tumor microenvironment (TME), may assist in the development of universal tumor targeting agents. The hypoxia-inducible factor (HIF), in particular HIF1, plays a key role in tumor adaptation to hypoxia, and inhibiting its interaction with p300 has been shown to provide therapeutic potential. Using a multivalent assembled protein (MAP) approach based on the self-assembly of the cartilage oligomeric matrix protein coiled-coil (COMPcc) domain fused to the critical residues of the C-terminal transactivation domain (C-TAD) of the α subunit of HIF1 (HIF1α), we generate HIF1α-MAP (H-MAP). The resulting H-MAP demonstrates picomolar binding affinity to p300, the ability to downregulate hypoxia-inducible genes, and in vivo tumor targeting capability.

Design of Coiled-Coil Protein Nanostructures for Therapeutics and Drug Delivery.

Coiled-coil protein motifs have become widely employed in the design of biomaterials. Some of these designs have been studied for use in drug delivery due to the unique ability of coiled-coils to impart stability, oligomerization, and supramolecular assembly. To leverage these properties and improve drug delivery, release, and targeting, a variety of nano- to mesoscale architectures have been adopted. Coiled-coil drug delivery and therapeutics have been developed by using the coiled-coil alone, designing for higher-order assemblies such as fibers and hydrogels, and combining coiled-coil proteins with other biocompatible structures such as lipids and polymers. We review the recent development of these structures and the design criteria used to generate functional proteins of varying sizes and morphologies.

Coiled-Coil Protein Hydrogels Engineered with Minimized Fiber Diameters for Sustained Release of Doxorubicin in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) lacks expressed protein targets, making therapy development challenging. Hydrogels offer a promising new route in this regard by improving the chemotherapeutic efficacy through increased solubility and sustained release. Moreover, subcutaneous hydrogel administration reduces patient burden by requiring less therapy and shorter treatment times. We recently established the design principles for the supramolecular assembly of single-domain coiled-coils into hydrogels. Using a modified computational design algorithm, we designed Q8, a hydrogel with rapid assembly for faster therapeutic hydrogel preparation. Q8 encapsulates and releases doxorubicin (Dox), enabling localized sustained release via subcutaneous injection. Remarkably, a single subcutaneous injection of Dox-laden Q8 (Q8•Dox) significantly suppresses tumors within just 1 week. This work showcases the bottom-up engineering of a fully protein-based drug delivery vehicle for improved TBNC treatment via noninvasive localized therapy.

Computational Design of Phosphotriesterase Improves V-Agent Degradation Efficiency.

Organophosphates (OPs) are a class of neurotoxic acetylcholinesterase inhibitors including widely used pesticides as well as nerve agents such as VX and VR. Current treatment of these toxins relies on reactivating acetylcholinesterase, which remains ineffective. Enzymatic scavengers are of interest for their ability to degrade OPs systemically before they reach their target. Here we describe a library of computationally designed variants of phosphotriesterase (PTE), an enzyme that is known to break down OPs. The mutations G208D, F104A, K77A, A80V, H254G, and I274N broadly improve catalytic efficiency of VX and VR hydrolysis without impacting the structure of the enzyme. The mutation I106 A improves catalysis of VR and L271E abolishes activity, likely due to disruptions of PTE's structure. This study elucidates the importance of these residues and contributes to the design of enzymatic OP scavengers with improved efficiency.

Supercharged coiled-coil protein with N-terminal decahistidine tag boosts siRNA complexation and delivery efficiency of a lipoproteoplex.

Short interfering RNA (siRNA) therapeutics have soared in popularity due to their highly selective and potent targeting of faulty genes, providing a non-palliative approach to address diseases. Despite their potential, effective transfection of siRNA into cells requires the assistance of an accompanying vector. Vectors constructed from non-viral materials, while offering safer and non-cytotoxic profiles, often grapple with lackluster loading and delivery efficiencies, necessitating substantial milligram quantities of expensive siRNA to confer the desired downstream effects. We detail the recombinant synthesis of a diverse series of coiled-coil supercharged protein (CSP) biomaterials systematically designed to investigate the impact of two arginine point mutations (Q39R and N61R) and decahistidine tags on liposomal siRNA delivery. The most efficacious variant, N8, exhibits a twofold increase in its affinity to siRNA and achieves a twofold enhancement in transfection activity with minimal cytotoxicity in vitro. Subsequent analysis unveils the destabilizing effect of the Q39R and N61R supercharging mutations and the incorporation of C-terminal decahistidine tags on α-helical secondary structure. Cross-correlational regression analyses reveal that the amount of helical character in these mutants is key in N8's enhanced siRNA complexation and downstream delivery efficiency.

Computational Prediction of Coiled-Coil Protein Gelation Dynamics and Structure.

Protein hydrogels represent an important and growing biomaterial for a multitude of applications, including diagnostics and drug delivery. We have previously explored the ability to engineer the thermoresponsive supramolecular assembly of coiled-coil proteins into hydrogels with varying gelation properties, where we have defined important parameters in the coiled-coil hydrogel design. Using Rosetta energy scores and Poisson-Boltzmann electrostatic energies, we iterate a computational design strategy to predict the gelation of coiled-coil proteins while simultaneously exploring five new coiled-coil protein hydrogel sequences. Provided this library, we explore the impact of in silico energies on structure and gelation kinetics, where we also reveal a range of blue autofluorescence that enables hydrogel disassembly and recovery. As a result of this library, we identify the new coiled-coil hydrogel sequence, Q5, capable of gelation within 24 h at 4 °C, a more than 2-fold increase over that of our previous iteration Q2. The fast gelation time of Q5 enables the assessment of structural transition in real time using small-angle X-ray scattering (SAXS) that is correlated to coarse-grained and atomistic molecular dynamics simulations revealing the supramolecular assembling behavior of coiled-coils toward nanofiber assembly and gelation. This work represents the first system of hydrogels with predictable self-assembly, autofluorescent capability, and a molecular model of coiled-coil fiber formation.

Protein-Engineered Fibers For Drug Encapsulation Traceable via 19F Magnetic Resonance.

Theranostic materials research is experiencing rapid growth driven by the interest in integrating both therapeutic and diagnostic modalities. These materials offer the unique capability to not only provide treatment but also track the progression of a disease. However, to create an ideal theranostic biomaterial without compromising drug encapsulation, diagnostic imaging must be optimized for improved sensitivity and spatial localization. Herein, we create a protein-engineered fluorinated coiled-coil fiber, Q2TFL, capable of improved sensitivity to 19F magnetic resonance spectroscopy (MRS) detection. Leveraging residue-specific noncanonical amino acid incorporation of trifluoroleucine (TFL) into the coiled-coil, Q2, which self-assembles into nanofibers, we generate Q2TFL. We demonstrate that fluorination results in a greater increase in thermostability and 19F magnetic resonance detection compared to the nonfluorinated parent, Q2. Q2TFL also exhibits linear ratiometric 19F MRS thermoresponsiveness, allowing it to act as a temperature probe. Furthermore, we explore the ability of Q2TFL to encapsulate the anti-inflammatory small molecule, curcumin (CCM), and its impact on the coiled-coil structure. Q2TFL also provides hyposignal contrast in 1H MRI, echogenic signal with high-frequency ultrasound and sensitive detection by 19F MRS in vivo illustrating fluorination of coiled-coils for supramolecular assembly and their use with 1H MRI, 19F MRS and high frequency ultrasound as multimodal theranostic agents.

Supramolecular Assembly and Small-Molecule Binding by Protein-Engineered Coiled-Coil Fibers.

The ability to engineer a solvent-exposed surface of self-assembling coiled coils allows one to achieve a higher-order hierarchical assembly such as nano- or microfibers. Currently, these materials are being developed for a range of biomedical applications, including drug delivery systems; however, ways to mechanistically optimize the coiled-coil structure for drug binding are yet to be explored. Our laboratory has previously leveraged the functional properties of the naturally occurring cartilage oligomeric matrix protein coiled coil (C), not only for its favorable motif but also for the presence of a hydrophobic pore to allow for small-molecule binding. This includes the development of Q, a rationally designed pentameric coiled coil derived from C. Here, we present a small library of protein microfibers derived from the parent sequences of C and Q bearing various electrostatic potentials with the aim to investigate the influence of higher-order assembly and encapsulation of candidate small molecule, curcumin. The supramolecular fiber size appears to be well-controlled by sequence-imbued electrostatic surface potential, and protein stability upon curcumin binding is well correlated to relative structure loss, which can be predicted by in silico docking.

Fluorinated Protein and Peptide Materials for Biomedical Applications.

Fluorination represents one of the most powerful modern design strategies to impart biomacromolecules with unique functionality, empowering them for widespread application in the biomedical realm. However, the properties of fluorinated protein materials remain unpredictable due to the heavy context-dependency of the surrounding atoms influenced by fluorine's strong electron-withdrawing tendencies. This review aims to discern patterns and elucidate design principles governing the biochemical synthesis and rational installation of fluorine into protein and peptide sequences for diverse biomedical applications. Several case studies are presented to deconvolute the overgeneralized fluorous stabilization effect and critically examine the duplicitous nature of the resultant enhanced chemical and thermostability as it applies to use as biomimetic therapeutics, drug delivery vehicles, and bioimaging modalities.

Engineered multivalent self-assembled binder protein against SARS-CoV-2 RBD.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global pandemic since December 2019, and with it, a push for innovations in rapid testing and neutralizing antibody treatments in an effort to solve the spread and fatality of the disease. One such solution to both of these prevailing issues is targeting the interaction of SARS-CoV-2 spike receptor binding domain (RBD) with the human angiotensin-converting enzyme 2 (ACE2) receptor protein. Structural studies have shown that the N-terminal alpha-helix comprised of the first 23 residues of ACE2 plays an important role in this interaction. Where it is typical to design a binding domain to fit a target, we have engineered a protein that relies on multivalency rather than the sensitivity of a monomeric ligand to provide avidity to its target by fusing the N-terminal helix of ACE2 to the coiled-coil domain of the cartilage oligomeric matrix protein. The resulting ACE-MAP is able to bind to the SARS-CoV-2 RBD with improved binding affinity, is expressible in E. coli, and is thermally stable and relatively small (62 kDa). These properties suggest ACE-MAP and the MAP scaffold to be a promising route towards developing future diagnostics and therapeutics to SARS-CoV-2.

Particle-Based Microrheology As a Tool for Characterizing Protein-Based Materials.

Microrheology based on video microscopy of embedded tracer particles has the potential to be used for high-throughput protein-based materials characterization. This potential is due to a number of characteristics of the techniques, including the suitability for measurement of low sample volumes, noninvasive and noncontact measurements, and the ability to set up a large number of samples for facile, sequential measurement. In addition to characterization of the bulk rheological properties of proteins in solution, for example, viscosity, microrheology can provide insight into the dynamics and self-assembly of protein-based materials as well as heterogeneities in the microenvironment being probed. Specifically, passive microrheology in the form of multiple particle tracking and differential dynamic microscopy holds promise for applications in high-throughput characterization because of the lack of user interaction required while making measurements. Herein, recent developments in the use of multiple particle tracking and differential dynamic microscopy are reviewed for protein characterization and their potential to be applied in a high-throughput, automatable setting.

Injectable recombinant block polymer gel for sustained delivery of therapeutic protein in post traumatic osteoarthritis.

Protein-based biomaterials offer several advantages over synthetic materials, owing to their unique stimuli-responsive properties, biocompatibility and modular nature. Here, we demonstrate that E5C, a recombinant protein block polymer, consisting of five repeats of elastin like polypeptide (E) and a coiled-coil domain of cartilage oligomeric matrix protein (C), is capable of forming a porous networked gel at physiological temperature, making it an excellent candidate for injectable biomaterials. Combination of E5C with Atsttrin, a chondroprotective engineered derivative of anti-inflammatory growth factor progranulin, provides a unique biochemical and biomechanical environment to protect against post-traumatic osteoarthritis (PTOA) onset and progression. E5C gel was demonstrated to provide prolonged release of Atsttrin and inhibit chondrocyte catabolism while facilitating anabolic signaling in vitro. We also provide in vivo evidence that prophylactic and therapeutic application of Atsttrin-loaded E5C gels protected against PTOA onset and progression in a rabbit anterior cruciate ligament transection model. Collectively, we have developed a unique protein-based gel capable of minimally invasive, sustained delivery of prospective therapeutics, particularly the progranulin-derivative Atsttrin, for therapeutic application in OA.

Protein Based Biomaterials for Therapeutic and Diagnostic Applications.

Proteins are some of the most versatile and studied macromolecules with extensive biomedical applications. The natural and biological origin of proteins offer such materials several advantages over their synthetic counterparts, such as innate bioactivity, recognition by cells and reduced immunogenic potential. Furthermore, proteins can be easily functionalized by altering their primary amino acid sequence and can often be further self-assembled into higher order structures either spontaneously or under specific environmental conditions. This review will feature the recent advances in protein-based biomaterials in the delivery of therapeutic cargo such as small molecules, genetic material, proteins, and cells. First, we will discuss the ways in which secondary structural motifs, the building blocks of more complex proteins, have unique properties that enable them to be useful for therapeutic delivery. Next, supramolecular assemblies, such as fibers, nanoparticles, and hydrogels, made from these building blocks that are engineered to behave in a cohesive manner, are discussed. Finally, we will cover additional modifications to protein materials that impart environmental responsiveness to materials. This includes the emerging field of protein molecular robots, and relatedly, protein-based theranostic materials that combine therapeutic potential with modern imaging modalities, including near-infrared fluorescence spectroscopy (NIRF), single-photo emission computed tomography/computed tomography (SPECT/CT), positron emission tomography (PET), magnetic resonance imaging (MRI), and ultrasound/photoacoustic imaging (US/PAI).

Engineered Protein-Iron Oxide Hybrid Biomaterial for MRI-traceable Drug Encapsulation.

Labeled protein-based biomaterials have become a popular for various biomedical applications such as tissue-engineered, therapeutic, or diagnostic scaffolds. Labeling of protein biomaterials, including with ultrasmall super-paramagnetic iron oxide (USPIO) nanoparticles, has enabled a wide variety of imaging techniques. These USPIO-based biomaterials are widely studied in magnetic resonance imaging (MRI), thermotherapy, and magnetically-driven drug delivery which provide a method for direct and non-invasive monitoring of implants or drug delivery agents. Where most developments have been made using polymers or collagen hydrogels, shown here is the use of a rationally designed protein as the building block for a meso-scale fiber. While USPIOs have been chemically conjugated to antibodies, glycoproteins, and tissue-engineered scaffolds for targeting or improved biocompatibility and stability, these constructs have predominantly served as diagnostic agents and often involve harsh conditions for USPIO synthesis. Here, we present an engineered protein-iron oxide hybrid material comprised of an azide-functionalized coiled-coil protein with small molecule binding capacity conjugated via bioorthogonal azide-alkyne cycloaddition to an alkyne-bearing iron oxide templating peptide, CMms6, for USPIO biomineralization under mild conditions. The coiled-coil protein, dubbed Q, has been previously shown to form nanofibers and, upon small molecule binding, further assembles into mesofibers via encapsulation and aggregation. The resulting hybrid material is capable of doxorubicin encapsulation as well as sensitive T2*-weighted MRI darkening for strong imaging capability that is uniquely derived from a coiled-coil protein.

Self-assembly of stimuli-responsive coiled-coil fibrous hydrogels.

Owing to their tunable properties, hydrogels comprised of stimuli-sensitive polymers are one of the most appealing scaffolds with applications in tissue engineering, drug delivery and other biomedical fields. We previously reported a thermoresponsive hydrogel formed using a coiled-coil protein, Q. Here, we expand our studies to identify the gelation of Q protein at distinct pH conditions, creating a protein hydrogel system that is sensitive to temperature and pH. Through secondary structure analysis, transmission electron microscopy, and rheology, we observed that Q self-assembles and forms fiber-based hydrogels exhibiting upper critical solution temperature behavior with increased elastic properties at pH 7.4 and pH 10. At pH 6, however, Q forms polydisperse nanoparticles, which do not further self-assemble and undergo gelation. The high net positive charge of Q at pH 6 creates significant electrostatic repulsion, preventing its gelation. This study will potentially guide the development of novel scaffolds and functional biomaterials that are sensitive towards biologically relevant stimuli.

Protein-based lateral flow assays for COVID-19 detection.

To combat the enduring and dangerous spread of COVID-19, many innovations to rapid diagnostics have been developed based on proteinprotein interactions of the SARS-CoV-2 spike and nucleocapsid proteins to increase testing accessibility. These antigen tests have most prominently been developed using the lateral flow assay (LFA) test platform which has the benefit of administration at point-of-care, delivering quick results, lower cost, and does not require skilled personnel. However, they have gained criticism for an inferior sensitivity. In the last year, much attention has been given to creating a rapid LFA test for detection of COVID-19 antigens that can address its high limit of detection while retaining the advantages of rapid antibodyantigen interaction. In this review, a summary of these proteinprotein interactions as well as the challenges, benefits, and recent improvements to protein based LFA for detection of COVID-19 are discussed.

Free-Standing Photocrosslinked Protein Polymer Hydrogels for Sustained Drug Release.

The fabrication of protein hydrogels consisting of different properties and functional motifs is critical in the development of protein-based materials for biomedical applications. Here, we report the design and characterization of a triblock protein polymer, CEC, composed of two different self-assembling domains derived from elastin protein (E) and coiled-coil protein (C), photopolymerized with a NHS-diazirine (D) crosslinker into a CEC-D hydrogel. The optimal photocrosslinker concentration and exposure time is determined to fabricate a free-standing hydrogel. Upon increasing the concentration of the CEC-D monomer and environmental temperature, the CEC-D hydrogel's conformation decreases in helical content from 58.0% to 44.8% and increases in ß-content from 25.9% to 38.1%. These gels experience 55 ± 6% protein erosion from the free-standing gel in 13 days as the gel films gradually decrease in size. The swelling ratio of 12 ± 1% denotes that the gel has a swelling ability comparable to other protein hydrogels. These photocrosslinked CEC-D hydrogels can be employed for drug delivery with high encapsulation and 14 ± 2% release of curcumin into the supernatant in a week long study. Overall, the photocrosslinked CEC-D hydrogels exhibit stability, swelling ability, and sustained release of drug.