Synthetic intrinsically disordered protein fusion tags that enhance protein solubility.

We report the de novo design of small (<20 kDa) and highly soluble synthetic intrinsically disordered proteins (SynIDPs) that confer solubility to a fusion partner with minimal effect on the activity of the fused protein. To identify highly soluble SynIDPs, we create a pooled gene-library utilizing a one-pot gene synthesis technology to create a large library of repetitive genes that encode SynIDPs. We identify three small (<20 kDa) and highly soluble SynIDPs from this gene library that lack secondary structure and have high solvation. Recombinant fusion of these SynIDPs to three known inclusion body forming proteins rescue their soluble expression and do not impede the activity of the fusion partner, thereby eliminating the need for removal of the SynIDP tag. These findings highlight the utility of SynIDPs as solubility tags, as they promote the soluble expression of proteins in E. coli and are small, unstructured proteins that minimally interfere with the biological activity of the fused protein.

Encoding Structure in Intrinsically Disordered Protein Biomaterials.

In nature, proteins range from those with highly ordered secondary and tertiary structures to those that completely lack a well-defined three-dimensional structure, termed intrinsically disordered proteins (IDPs). IDPs are generally characterized by one or more segments that have a compositional bias toward small hydrophilic amino acids and proline residues that promote structural disorder and are called intrinsically disordered regions (IDRs). The combination of IDRs with ordered regions and the interactions between the two determine the phase behavior, structure, and function of IDPs. Nature also diversifies the structure of proteins and thereby their functions by hybridization of the proteins with other moieties such as glycans and lipids; for instance, post-translationally glycosylated and lipidated proteins are important cell membrane components. Additionally, diversity in protein structure and function is achieved in nature through cross-linking proteins within themselves or with other domains to create various topologies. For example, an essential characteristic of the extracellular matrix (ECM) is the cross-linking of its network components, including proteins such as collagen and elastin, as well as polysaccharides such as hyaluronic acid (HA). Inspired by nature, synthetic IDP (SynIDP)-based biomaterials can be designed by employing similar strategies with the goal of introducing structural diversity and hence unique physiochemical properties. This Account describes such materials produced over the past decade and following one or more of the following approaches: (1) incorporating highly ordered domains into SynIDPs, (2) conjugating SynIDPs to other moieties through either genetically encoded post-translational modification or chemical conjugation, and (3) engineering the topology of SynIDPs via chemical modification. These approaches introduce modifications to the primary structure of SynIDPs, which are then translated to unique three-dimensional secondary and tertiary structures. Beginning with completely disordered SynIDPs as the point of origin, structure may be introduced into SynIDPs by each of these three unique approaches individually along orthogonal axes or by combinations of the three, enabling bioinspired designs to theoretically span the entire range of three-dimensional structural possibilities. Furthermore, the resultant structures span a wide range of length scales, from nano- to meso- to micro- and even macrostructures. In this Account, emphasis is placed on the physiochemical properties and structural features of the described materials. Conjugates of SynIDPs to synthetic polymers and materials achieved by simple mixing of components are outside the scope of this Account. Related biomedical applications are described briefly. Finally, we note future directions for the design of functional SynIDP-based biomaterials.

Modulating hierarchical self-assembly in thermoresponsive intrinsically disordered proteins through high-temperature incubation time.

The cornerstone of structural biology is the unique relationship between protein sequence and the 3D structure at equilibrium. Although intrinsically disordered proteins (IDPs) do not fold into a specific 3D structure, breaking this paradigm, some IDPs exhibit large-scale organization, such as liquid-liquid phase separation. In such cases, the structural plasticity has the potential to form numerous self-assembled structures out of thermal equilibrium. Here, we report that high-temperature incubation time is a defining parameter for micro and nanoscale self-assembly of resilin-like IDPs. Interestingly, high-resolution scanning electron microscopy micrographs reveal that an extended incubation time leads to the formation of micron-size rods and ellipsoids that depend on the amino acid sequence. More surprisingly, a prolonged incubation time also induces amino acid composition-dependent formation of short-range nanoscale order, such as periodic lamellar nanostructures. We, therefore, suggest that regulating the period of high-temperature incubation, in the one-phase regime, can serve as a unique method of controlling the hierarchical self-assembly mechanism of structurally disordered proteins.

Potent inhibition of MMP-9 by a novel sustained-release platform attenuates left ventricular remodeling following myocardial infarction.

Sustained drug-release systems prolong the retention of therapeutic drugs within target tissues to alleviate the need for repeated drug administration. Two major caveats of the current systems are that the release rate and the timing cannot be predicted or fine-tuned because they rely on uncontrolled environmental conditions and that the system must be redesigned for each drug and treatment regime because the drug is bound via interactions that are specific to its structure and composition. We present a controlled and universal sustained drug-release system, which comprises minute spherical particles in which a therapeutic protein is affinity-bound to alginate sulfate (AlgS) through one or more short heparin-binding peptide (HBP) sequence repeats. Employing post-myocardial infarction (MI) heart remodeling as a case study, we show that the release of C9-a matrix metalloproteinase-9 (MMP-9) inhibitor protein that we easily bound to AlgS by adding one, two, or three HBP repeats to its sequence-can be directly controlled by modifying the number of HBP repeats. In an in vivo study, we directly injected AlgS particles, which were bound to C9 through three HBP repeats, into the left ventricular myocardium of mice following MI. We found that the particles substantially reduced post-MI remodeling, attesting to the sustained, local release of the drug within the tissue. As the number of HBP repeats controls the rate of drug release from the AlgS particles, and since C9 can be easily replaced with almost any protein, our tunable sustained-release system can readily accommodate a wide range of protein-based treatments.

Modulating Hierarchical Self-Assembly In Thermoresponsive Intrinsically Disordered Proteins Through High-Temperature Incubation Time.

The cornerstone of structural biology is the unique relationship between protein sequence and the 3D structure at equilibrium. Although intrinsically disordered proteins (IDPs) do not fold into a specific 3D structure, breaking this paradigm, some IDPs exhibit large-scale organization, such as liquid-liquid phase separation. In such cases, the structural plasticity has the potential to form numerous self-assembled structures out of thermal equilibrium. Here, we report that high-temperature incubation time is a defining parameter for micro and nanoscale self-assembly of resilin-like IDPs. Interestingly, high-resolution scanning electron microscopy micrographs reveal that an extended incubation time leads to the formation of micron-size rods and ellipsoids that depend on the amino acid sequence. More surprisingly, a prolonged incubation time also induces amino acid composition-dependent formation of short-range nanoscale order, such as periodic lamellar nanostructures. We can correlate the lamellar structures to \b{eta}-sheet formation and demonstrate similarities between the observed nanoscopic structural arrangement and spider silk. We, therefore, suggest that regulating the period of high-temperature incubation, in the one-phase regime, can serve as a unique method of controlling the hierarchical self-assembly mechanism of structurally disordered proteins.

Modulating Hierarchical Self-Assembly In Thermoresponsive Intrinsically Disordered Proteins Through High-Temperature Incubation Time.

The cornerstone of structural biology is the unique relationship between protein sequence and the 3D structure at equilibrium. Although intrinsically disordered proteins (IDPs) do not fold into a specific 3D structure, breaking this paradigm, some IDPs exhibit large-scale organization, such as liquid-liquid phase separation. In such cases, the structural plasticity has the potential to form numerous self-assembled structures out of thermal equilibrium. Here, we report that high-temperature incubation time is a defining parameter for micro and nanoscale self-assembly of resilin-like IDPs. Interestingly, high-resolution scanning electron microscopy micrographs reveal that an extended incubation time leads to the formation of micron-size rods and ellipsoids that depend on the amino acid sequence. More surprisingly, a prolonged incubation time also induces amino acid composition-dependent formation of short-range nanoscale order, such as periodic lamellar nanostructures. We can correlate the lamellar structures to ß-sheet formation and demonstrate similarities between the observed nanoscopic structural arrangement and spider silk. We, therefore, suggest that regulating the period of high-temperature incubation, in the one-phase regime, can serve as a unique method of controlling the hierarchical self-assembly mechanism of structurally disordered proteins.

APPI-Derived Cyclic Peptide Enhances Aß42 Aggregation and Reduces Aß42-Mediated Membrane Destabilization and Cytotoxicity.

An amyloid precursor protein inhibitor (APPI) and amyloid beta 42 (Aß42) are both subdomains of the human transmembrane amyloid precursor protein (APP). In the brains of patients with Alzheimer's disease (AD), Aß42 oligomerizes into aggregates of various sizes, with intermediate, low-molecular-weight Aß42 oligomers currently being held to be the species responsible for the most neurotoxic effects associated with the disease. Strategies to ameliorate the toxicity of these intermediate Aß42 oligomeric species include the use of short, Aß42-interacting peptides that either inhibit the formation of the Aß42 oligomeric species or promote their conversion to high-molecular-weight aggregates. We therefore designed such an Aß42-interacting peptide that is based on the ß-hairpin amino acid sequence of the APPI, which exhibits high similarity to the ß-sheet-like aggregation site of Aß42. Upon tight binding of this 20-mer cyclic peptide to Aß42 (in a 1:1 molar ratio), the formation of Aß42 aggregates was enhanced, and consequently, Aß42-mediated cell toxicity was ameliorated. We showed that in the presence of the cyclic peptide, interactions of Aß42 with both plasma and mitochondrial membranes and with phospholipid vesicles that mimic these membranes were inhibited. Specifically, the cyclic peptide inhibited Aß42-mediated mitochondrial membrane depolarization and reduced Aß42-mediated apoptosis and cell death. We suggest that the cyclic peptide modulates Aß42 aggregation by enhancing the formation of large aggregates─as opposed to low-molecular-weight intermediates─and as such has the potential for further development as an AD therapeutic.

Activating hidden signals by mimicking cryptic sites in a synthetic extracellular matrix.

Cryptic sites are short signaling peptides buried within the native extracellular matrix (ECM). Enzymatic cleavage of an ECM protein reveals these hidden peptide sequences, which interact with surface receptors to control cell behavior. Materials that mimic this dynamic interplay between cells and their surroundings via cryptic sites could enable application of this endogenous signaling phenomenon in synthetic ECM hydrogels. We demonstrate that depsipeptides ("switch peptides") can undergo enzyme-triggered changes in their primary sequence, with proof-of-principle studies showing how trypsin-triggered primary sequence rearrangement forms the bioadhesive pentapeptide YIGSR. We then engineered cryptic site-mimetic synthetic ECM hydrogels that experienced a cell-initiated gain of bioactivity. Responding to the endothelial cell surface enzyme aminopeptidase N, the inert matrix transformed into an adhesive synthetic ECM capable of supporting endothelial cell growth. This modular system enables dynamic reciprocity in synthetic ECMs, reproducing the natural symbiosis between cells and their matrix through inclusion of tunable hidden signals.

Surfactant-Mediated Co-Existence of Single-Walled Carbon Nanotube Networks and Cellulose Nanocrystal Mesophases.

Hybrids comprising cellulose nanocrystals (CNCs) and percolated networks of single-walled carbon nanotubes (SWNTs) may serve for the casting of hybrid materials with improved optical, mechanical, electrical, and thermal properties. However, CNC-dispersed SWNTs are depleted from the chiral nematic (N*) phase and enrich the isotropic phase. Herein, we report that SWNTs dispersed by non-ionic surfactant or triblock copolymers are incorporated within the surfactant-mediated CNC mesophases. Small-angle X-ray measurements indicate that the nanostructure of the hybrid phases is only slightly modified by the presence of the surfactants, and the chiral nature of the N* phase is preserved. Cryo-TEM and Raman spectroscopy show that SWNTs networks with typical mesh size from hundreds of nanometers to microns are distributed equally between the two phases. We suggest that the adsorption of the surfactants or polymers mediates the interfacial interaction between the CNCs and SWNTs, enhancing the formation of co-existing meso-structures in the hybrid phases.

Avidity observed between a bivalent inhibitor and an enzyme monomer with a single active site.

Although myriad protein-protein interactions in nature use polyvalent binding, in which multiple ligands on one entity bind to multiple receptors on another, to date an affinity advantage of polyvalent binding has been demonstrated experimentally only in cases where the target receptor molecules are clustered prior to complex formation. Here, we demonstrate cooperativity in binding affinity (i.e., avidity) for a protein complex in which an engineered dimer of the amyloid precursor protein inhibitor (APPI), possessing two fully functional inhibitory loops, interacts with mesotrypsin, a soluble monomeric protein that does not self-associate or cluster spontaneously. We found that each inhibitory loop of the purified APPI homodimer was over three-fold more potent than the corresponding loop in the monovalent APPI inhibitor. This observation is consistent with a suggested mechanism whereby the two APPI loops in the homodimer simultaneously and reversibly bind two corresponding mesotrypsin monomers to mediate mesotrypsin dimerization. We propose a simple model for such dimerization that quantitatively explains the observed cooperativity in binding affinity. Binding cooperativity in this system reveals that the valency of ligands may affect avidity in protein-protein interactions including those of targets that are not surface-anchored and do not self-associate spontaneously. In this scenario, avidity may be explained by the enhanced concentration of ligand binding sites in proximity to the monomeric target, which may favor rebinding of the multiple ligand binding sites with the receptor molecules upon dissociation of the protein complex.

Crescent-Shaped Supramolecular Tetrapeptide Nanostructures.

Self-assembly of amphiphilic peptide-based building blocks gives rise to a plethora of interesting nanostructures such as ribbons, fibers, and tubes. However, it remains a great challenge to employ peptide self-assembly to directly produce nanostructures with lower symmetry than these highly symmetric motifs. We report here our discovery that persistent and regular crescent nanostructures with a diameter of 28 ± 3 nm formed from a series of tetrapeptides with the general structure AdKSKSEX (Ad = adamantyl group, KS = lysine residue functionalized with an S-aroylthiooxime (SATO) group, E = glutamic acid residue, and X = variable amino acid residue). In the presence of cysteine, the biological signaling gas hydrogen sulfide (H2S) was released from the SATO units of the crescent nanostructures, termed peptide-H2S donor conjugates (PHDCs), reducing levels of reactive oxygen species (ROS) in macrophage cells. Additional in vitro studies showed that the crescent nanostructures alleviated cytotoxicity induced by phorbol 12-myristate-13-acetate more effectively than common H2S donors and a PHDC of a similar chemical structure, AdKSKSE, that formed short nanoworms instead of nanocrescents. Cell internalization studies indicated that nanocrescent-forming PHDCs were more effective in reducing ROS levels in macrophages because they entered into and remained in cells better than nanoworms, highlighting how nanostructure morphology can affect bioactivity in drug delivery.

A combined experimental and computational approach reveals how aromatic peptide amphiphiles self-assemble to form ion-conducting nanohelices.

Reported here is a combined experimental-computational strategy to determine structure-property-function relationships in persistent nanohelices formed by a set of aromatic peptide amphiphile (APA) tetramers with the general structure K S XEK S , where KS= S-aroylthiooxime modified lysine, X = glutamic acid or citrulline, and E = glutamic acid. In low phosphate buffer concentrations, the APAs self-assembled into flat nanoribbons, but in high phosphate buffer concentrations they formed nanohelices with regular twisting pitches ranging from 9-31 nm. Coarse-grained molecular dynamics simulations mimicking low and high salt concentrations matched experimental observations, and analysis of simulations revealed that increasing strength of hydrophobic interactions under high salt conditions compared with low salt conditions drove intramolecular collapse of the APAs, leading to nanohelix formation. Analysis of the radial distribution functions in the final self-assembled structures led to several insights. For example, comparing distances between water beads and beads representing hydrolysable KS units in the APAs indicated that the KS units in the nanohelices should undergo hydrolysis faster than those in the nanoribbons; experimental results verified this hypothesis. Simulation results also suggested that these nanohelices might display high ionic conductivity due to closer packing of carboxylate beads in the nanohelices than in the nanoribbons. Experimental results showed no conductivity increase over baseline buffer values for unassembled APAs, a slight increase (0.4 × 102 µS/cm) for self-assembled APAs under low salt conditions in their nanoribbon form, and a dramatic increase (8.6 × 102 µS/cm) under high salt conditions in their nanohelix form. Remarkably, under the same salt conditions, these self-assembled nanohelices conducted ions 5-10-fold more efficiently than several charged polymers, including alginate and DNA. These results highlight how experiments and simulations can be combined to provide insight into how molecular design affects self-assembly pathways; additionally, this work highlights how this approach can lead to discovery of unexpected properties of self-assembled nanostructures.

Effect of Crosslinker Topology on Enzymatic Degradation of Hydrogels.

Despite the widespread use of hydrogels in biomedical applications, little is known regarding the effect of crosslinker topology on hydrogel degradation. Dendritic and linear elastin-like peptides (ELPs) were used as crosslinkers for hyaluronic acid (HA) hydrogels, and their enzymatic degradation was studied using trypsin. Rheological studies revealed that hydrogels crosslinked with ELP dendrimers (HA_denELPs) degraded more slowly than those crosslinked with the otherwise equivalent linear ELPs (i.e., both molecules have the same number of pentamers and peripheral lysine residues). The origin of this phenomenon was evaluated using solution studies in which various dendritic and linear ELPs were treated with trypsin. Apart from the expected steric hindrances due to the dendritic topology, we identified the dual directionality of the peptide sequences (generated by a central branching lysine residue) and the likelihood of cleaving a productive crosslinking point as two additional contributors to the lesser degradability of HA_denELPs. Overall, these results highlight how the molecular design of crosslinker topology represents a novel strategy to tune the degradation rate of hydrogels.

Hydrogels composed of hyaluronic acid and dendritic ELPs: hierarchical structure and physical properties.

Hydrogels that mimic the native extracellular matrix were prepared from hyaluronic acid (HA) and amine-terminated dendritic elastin-like peptides (denELPs) of generations 1, 2, and 3 (G1, 2, and 3) as crosslinking units. The physical properties of the hydrogels were investigated by rheology, scanning electron microscopy, swelling tests, small-angle X-ray scattering (SAXS), and model drug loading and release assays. Hydrogel properties depended on the generation number of the denELP, which contained structural segments based on the repeating GLPGL pentamer. Hydrogels with higher generation denELPs (G2 and 3) showed similar properties, but those prepared from G1 denELPs were rheologically weaker, had a larger mesh size, absorbed less model drug, and released the drug more quickly. Interestingly, most of the HA_denELP hydrogels studied here remained transparent upon gelation, but after lyophilization and addition of water retained opaque, "solid-like" regions for up to 4 d during rehydration. This rehydration process was carefully evaluated through time-course SAXS studies, and the phenomenon was attributed to the formation of pre-coacervates in the gel-forming step, which slowly swelled in water during rehydration. These findings provide important insights into the behavior of ELP-based hydrogels, in which physical crosslinking of the ELP domains can be controlled to tune mechanical properties, highlighting the potential of HA_denELP hydrogels as biomaterials.

Oligomerization and Auto-methylation of the Human Lysine Methyltransferase SETD6.

Signaling via lysine methylation by protein lysine methyltransferases (PKMTs), has been linked to diverse biological and disease processes. The mono-methyltransferase SETD6 (SET-domain-containing protein 6) is a member of the PKMT family and was previously shown to regulate essential cellular processes such as the NF-κB, WNT and the oxidative stress pathways. However, on the biochemical level, little is known about the enzymatic mode of action of SETD6. Here we provide evidence that SETD6 forms high-molecular-weight structures. Specifically, we demonstrate that SETD6 monomeric, dimeric and trimeric forms are stabilized by the methyl donor, S-adenosyl-l-methionine. We then show that SETD6 has auto-methylation activity at K39 and K179, which serves as the major auto-methylation sites with a moderate auto-methylation activity toward K372. A point mutation at K179 but not at K39 and K372, located at the SET domain of SETD6, impaired SETD6 ability to form a trimer, strongly implying a link between the auto-methylation and the oligomerization state. Finally, by radioactive in vitro methylation experiments and biochemical kinetics analysis, we show that the auto-methylation at K39 and K179 increases the catalytic rate of SETD6. Collectively, our data support a model by which SETD6 auto-methylation and self-interaction positively regulate its enzymatic activity in vitro and may suggest that other PKMTs are regulated in the same manner.

Multi-scale characterization of thermoresponsive dendritic elastin-like peptides.

Elastin like peptides (ELPs)-polypeptides based on the protein elastin-are used widely as thermoresponsive components in biomaterials due to the presence of a sharp soluble-to-insoluble phase change at a characteristic transition temperature (Tt). While linear ELPs have been thoroughly studied, few investigations into branched ELPs have been carried out. Using lysine amino acids as branching and terminal units with 1-3 pentameric repeats between each branch, ELP dendrimers were prepared by solid-phase peptide synthesis with molecular weights as high as 14kDa. A conformation change from random coil to ß-turn upon heating through the Tt, typical of ELPs, was observed by circular dichroism spectroscopy for all peptides. The high molecular weights of these peptides enabled the use of characterization techniques typically reserved for polymers. Variable-temperature small-angle X-ray scattering measurements in dilute solution revealed an increase in size and fractal dimension upon heating, even well below the Tt. These results were corroborated by cryogenic transmission electron microscopy, which confirmed the presence of aggregates below the Tt, and micro differential scanning calorimetry, which showed a broad endothermic peak below the Tt. These results collectively indicate the presence of a pre-coacervation step in the phase transition of ELP dendrimers.