Precision Loading and Delivery of Molecular Cargo by Size-Controlled Coacervation of Gold Nanoparticles Functionalized with Elastin-like Peptides.

Thermoresponsive elastin-like peptides (ELPs) have been extensively investigated in biotechnology and medicine, but little attention has been paid to the process by which coacervation causes ELP-decorated particles to aggregate. Using gold nanoparticles (AuNPs) functionalized with a cysteine-terminated 96-repeat of the VPGVG sequence (V96-Cys), we show that the size of the clusters that reversibly form above the ELP transition temperature can be finely controlled in the 250 to 930 nm range by specifying the concentration of free V96-Cys in solution and using AuNPs of different sizes. We further find that the localized surface plasmon resonance peak of the embedded AuNPs progressively red-shifts with cluster size, likely due to an increase in particle-particle contacts. We exploit this fine control over size to homogeneously load precise amounts of the dye Nile Red and the antibiotic Tetracycline into clusters of different hydrodynamic diameters and deliver cargos near-quantitatively by deconstructing the aggregates below the ELP transition temperature. Beyond establishing a key role for free ELPs in the agglomeration of ELP-functionalized particles, our results provide a path for the thermally controlled delivery of precise quantities of molecular cargo. This capability might prove useful in combination photothermal therapies and theranostic applications, and to trigger spatially and temporally uniform responses from biological, electronic, or optical systems.

Towards predictive control of reversible nanoparticle assembly with solid-binding proteins.

Although a broad range of ligand-functionalized nanoparticles and physico-chemical triggers have been exploited to create stimuli-responsive colloidal systems, little attention has been paid to the reversible assembly of unmodified nanoparticles with non-covalently bound proteins. Previously, we reported that a derivative of green fluorescent protein engineered with oppositely located silica-binding peptides mediates the repeated assembly and disassembly of 10-nm silica nanoparticles when pH is toggled between 7.5 and 8.5. We captured the subtle interplay between interparticle electrostatic repulsion and their protein-mediated short-range attraction with a multiscale model energetically benchmarked to collective system behavior captured by scattering experiments. Here, we show that both solution conditions (pH and ionic strength) and protein engineering (sequence and position of engineered silica-binding peptides) provide pathways for reversible control over growth and fragmentation, leading to clusters ranging in size from 25 nm protein-coated particles to micrometer-size aggregate. We further find that the higher electrolyte environment associated with successive cycles of base addition eventually eliminates reversibility. Our model accurately predicts these multiple length scales phenomena. The underpinning concepts provide design principles for the dynamic control of other protein- and particle-based nanocomposites.

Controlling Mineralization with Protein-Functionalized Peptoid Nanotubes.

Sequence-defined foldamers that self-assemble into well-defined architectures are promising scaffolds to template inorganic mineralization. However, it has been challenging to achieve robust control of nucleation and growth without sequence redesign or extensive experimentation. Here, peptoid nanotubes functionalized with a panel of solid-binding proteins are used to mineralize homogeneously distributed and monodisperse anatase nanocrystals from the water-soluble TiBALDH precursor. Crystallite size is systematically tuned between 1.4 and 4.4 nm by changing protein coverage and the identity and valency of the genetically engineered solid-binding segments. The approach is extended to the synthesis of gold nanoparticles and, using a protein encoding both material-binding specificities, to the fabrication of titania/gold nanocomposites capable of photocatalysis under visible-light illumination. Beyond uncovering critical roles for hierarchical organization and denticity on solid-binding protein mineralization outcomes, the strategy described herein should prove valuable for the fabrication of hierarchical hybrid materials incorporating a broad range of inorganic components.

Hierarchical Materials from High Information Content Macromolecular Building Blocks: Construction, Dynamic Interventions, and Prediction.

Hierarchical materials that exhibit order over multiple length scales are ubiquitous in nature. Because hierarchy gives rise to unique properties and functions, many have sought inspiration from nature when designing and fabricating hierarchical matter. More and more, however, nature's own high-information content building blocks, proteins, peptides, and peptidomimetics, are being coopted to build hierarchy because the information that determines structure, function, and interfacial interactions can be readily encoded in these versatile macromolecules. Here, we take stock of recent progress in the rational design and characterization of hierarchical materials produced from high-information content blocks with a focus on stimuli-responsive and "smart" architectures. We also review advances in the use of computational simulations and data-driven predictions to shed light on how the side chain chemistry and conformational flexibility of macromolecular blocks drive the emergence of order and the acquisition of hierarchy and also on how ionic, solvent, and surface effects influence the outcomes of assembly. Continued progress in the above areas will ultimately usher in an era where an understanding of designed interactions, surface effects, and solution conditions can be harnessed to achieve predictive materials synthesis across scale and drive emergent phenomena in the self-assembly and reconfiguration of high-information content building blocks.

Peptoid-Directed Formation of Five-Fold Twinned Au Nanostars through Particle Attachment and Facet Stabilization.

While bio-inspired synthesis offers great potential for controlling nucleation and growth of inorganic particles, precisely tuning biomolecule-particle interactions is a long-standing challenge. Herein, we used variations in peptoid sequence to manipulate peptoid-Au interactions, leading to the synthesis of concave five-fold twinned, five-pointed Au nanostars via a process of repeated particle attachment and facet stabilization. Ex situ and liquid-phase TEM observations show that a balance between particle attachment biased to occur near the star points, preferential growth along the [100] direction, and stabilization of (111) facets is critical to forming star-shaped particles. Molecular simulations predict that interaction strengths between peptoids and distinct Au facets differ significantly and thus can alter attachment kinetics and surface energies to form the stars. This work provides new insights into how sequence-defined ligands affect particle growth to regulate crystal morphology.

Predictive Theoretical Framework for Dynamic Control of Bioinspired Hybrid Nanoparticle Self-Assembly.

At-will tailoring of the formation and reconfiguration of hierarchical structures is a key goal of modern nanomaterial design. Bioinspired systems comprising biomacromolecules and inorganic nanoparticles have potential for new functional material structures. Yet, consequential challenges remain because we lack a detailed understanding of the temporal and spatial interplay between participants when it is mediated by fundamental physicochemical interactions over a wide range of scales. Motivated by a system in which silica nanoparticles are reversibly and repeatedly assembled using a homobifunctional solid-binding protein and single-unit pH changes under near-neutral solution conditions, we develop a theoretical framework where interactions at the molecular and macroscopic scales are rigorously coupled based on colloidal theory and atomistic molecular dynamics simulations. We integrate these interactions into a predictive coarse-grained model that captures the pH-dependent reversibility and accurately matches small-angle X-ray scattering experiments at collective scales. The framework lays a foundation to connect microscopic details with the macroscopic behavior of complex bioinspired material systems and to control their behavior through an understanding of both equilibrium and nonequilibrium characteristics.

Interrogating biomineralization one amino acid at a time: amplification of mutational effects in protein-aided titania morphogenesis through reaction-diffusion control.

To emulate the control that biomineralizing organisms exert over reactant transport, we construct a countercurrent reaction-diffusion chamber in which an agarose hydrogel regulates the fluxes of inorganic precursor and precipitating solid-binding protein. We show that the morphology of the bioprecipitated titania can be changed from monolithic to interconnected particle networks and dispersed nanoparticles either by decreasing reaction time or by increasing agarose weight percentage at constant precursor and protein concentrations. More strikingly, protein variants with one or two substitutions in their metal oxide-binding domain yield unique peripheral morphologies (needles, threads, plates, and peapods) with distinct crystallography and photocatalytic activity. Our results suggest that diffusional control can magnify otherwise subtle mutational effects in biomineralizing proteins and provide a path for the green synthesis of morphologically and functionally diverse inorganic materials.

Solid-Binding Proteins: Bridging Synthesis, Assembly, and Function in Hybrid and Hierarchical Materials Fabrication.

There is considerable interest in the development of hybrid organic-inorganic materials because of the potential for harvesting the unique capabilities that each system has to offer. Proteins are an especially attractive organic component owing to the high amount of chemical information encoded in their amino acid sequence, their amenability to molecular and computational (re)design, and the many structures and functions they specify. Genetic installation of solid-binding peptides (SBPs) within protein frameworks affords control over the position and orientation of adhesive and morphogenetic segments, and a path toward predictive synthesis and assembly of functional materials and devices, all while harnessing the built-in properties of the host scaffold. Here, we review the current understanding of the mechanisms through which SBPs bind to technologically relevant interfaces, with an emphasis on the variables that influence the process, and highlight the last decade of progress in the use of solid-binding proteins for hybrid and hierarchical materials synthesis.

Nanoparticle-Mediated Assembly of Peptoid Nanosheets Functionalized with Solid-Binding Proteins: Designing Heterostructures for Hierarchy.

The fabrication of ordered architectures that intimately integrate polymer, protein, and inorganic components remains difficult. Two promising building blocks to tackle this challenge are peptoids, peptide mimics capable of self-assembly into well-defined structures, and solid-binding peptides, which offer a biological path to controlled inorganic assembly. Here, we report on the synthesis of 3.3-nm-thick thiol-reactive peptoid nanosheets from equimolar mixtures of unmodified and maleimide-derivatized versions of the Nbpe6Nce6 oligomer, optimize the location of engineered cysteine residues in silica-binding derivatives of superfolder green fluorescent protein for maleimide conjugation, and react the two components to form protein-peptoid hybrids exhibiting partial or uniform protein coverage on both of their sides. Using 10 nm silica nanoparticles, we trigger the stacking of these 2D structures into a multilayered material composed of alternating peptoid, protein, and organic layers. This simple and modular approach to hierarchical hybrid synthesis should prove useful in bioimaging and photocatalysis applications.

Phase Control of Nanocrystalline Inclusions in Bioprecipitated Titania with a Panel of Mutant Silica-Binding Proteins.

The biomimetic route to inorganic synthesis presents an opportunity to produce complex materials with superior properties under ambient conditions and from nontoxic precursors. While there has been significant progress in using solid-binding peptides (SBPs), proteins, and organisms to produce a variety of inorganic and hybrid structures, it has been more challenging to understand the interplay of solution conditions and solid-binding peptide (SBP) sequence, structure, and self-association on synthetic outcomes. Here, we show that fusing the Car9 silica-binding peptide-but not the silaffin-derived R5 peptide-to superfolder green fluorescent protein (sfGFP) enhances the ability of micromolar concentrations of protein to induce rapid titania (TiO2) precipitation from acidified solutions of tetrakis(di-lactato)-oxo-titanate (TiBALDH). TiO2 is produced stoichiometrically and although predominantly amorphous, contains nanosized anatase and monoclinic TiO2(B) inclusions. Remarkably, the phase of these nanocrystallites can be tuned from about 80% TiO2(B) to about 65% anatase by using Car9 mutants impaired in their ability to drive the formation of higher-order sfGFP-Car9 oligomers. Our results suggest that the presentation of multiple basic side chains in an extended plane formed by SBP self-association is critical to template the formation of monoclinic crystallites and underscore the subtle influence that single or dual substitutions in dodecameric SBPs can exert on the yield and crystallinity of biomineralized inorganics.

Affinity purification of Car9-tagged proteins on silica-derivatized spin columns and 96-well plates.

The Car9 affinity tag is a dodecameric silica-binding peptide that can be fused to the N- and C-termini of proteins of interest to enable their rapid and inexpensive purification on underivatized silica in a process that typically relies on l-lysine as an eluent. Here, we show that silica paper spin columns and borosilicate multi-well plates used for plasmid DNA purification are suitable for recovering Car9-tagged proteins with high purity in a workflow compatible with high-throughput experiments. Spin columns typically yield 100 µg of biologically active material that can be recovered in minutes with low concentrations of lysine. Because of their short bed length, spin columns also offer unique advantages, as evidenced by the selective recovery of functional Car9-tagged tobacco etch virus (TEV) protease from a fused and auto-cleaved maltose binding protein (MBP) folding partner that nonspecifically binds to silica in the presence of NaCl. These additional purification modalities should increase the versatility and appeal of the Car9 tag for affinity protein purification.

Sequence-Structure-Binding Relationships Reveal Adhesion Behavior of the Car9 Solid-Binding Peptide: An Integrated Experimental and Simulation Study.

Solid-binding peptides (SBPs) recognizing inorganic and synthetic interfaces have enabled a broad range of materials science applications and hold promise as adhesive or morphogenetic control units that can be genetically encoded within desirable or designed protein frameworks. To date, the underlying relationships governing both SBP-surface and SBP-SBP interactions and how they give rise to different adsorption mechanisms remain unclear. Here, we combine protein engineering, surface plasmon resonance characterization, and molecular dynamics (MD) simulations initiated from Rosetta predictions to gain insights on the interplay of amino acid composition, structure, self-association, and adhesion modality in a panel of variants of the Car9 silica-binding peptide (DSARGFKKPGKR) fused to the C-terminus of superfolder green fluorescent protein (sfGFP). Analysis of kinetics, energetics, and MD-predicted structures shows that the high-affinity binding of Car9 to the silanol-rich surface of silica is dominated by electrostatic contributions and a spectrum of several persistent interactions that, along with a high surface population of bound molecules, promote cooperative interactions between neighboring SBPs and higher order structure formation. Transition from cooperative to Langmuir adhesion in sfGFP-Car9 variants occurs in concert with a reduction of stable surface interactions and self-association, as confirmed by atomic force microscopy imaging of proteins exhibiting the two different binding behaviors. We discuss the implications of these results for the de novo design of SBP-surface binding systems.

Modeling the Cooperative Adsorption of Solid-Binding Proteins on Silica: Molecular Insights from Surface Plasmon Resonance Measurements.

Combinatorially selected solid-binding peptides (SBPs) provide a versatile route for synthesizing advanced materials and devices, especially when they are installed within structurally or functionally useful protein scaffolds. However, their promise has not been fully realized because we lack a predictive understanding of SBP-material interactions. Thermodynamic and kinetic binding parameters obtained by fitting quartz crystal microbalance and surface plasmon resonance (SPR) data with the Langmuir model whose assumptions are rarely satisfied provide limited information on underpinning molecular interactions. Using SPR, we show here that a technologically useful SBP called Car9 confers proteins to which is fused a sigmoidal adsorption behavior modulated by partner identity, quaternary structure, and ionic strength. We develop a two-step cooperative model that accurately captures the kinetics of silica binding and provides insights into how SBP-SBP interactions, fused scaffold, and solution conditions modulate adsorption. Because cooperative binding can be converted to Langmuir adhesion by mutagenesis, our approach offers a path to identify and to better understand and design practically useful SBPs.

Selective Labeling and Decoration of the Ends and Sidewalls of Single-Walled Carbon Nanotubes Using Mono- and Bispecific Solid-Binding Fluorescent Proteins.

Simple and robust strategies for the noncovalent functionalization of carbon nanostructures with proteins are of considerable interest in hybrid nanomaterials synthesis, part-to-part assembly, and biosensor development. Here, we show that fusion of the Car9 and Car15 carbon-binding peptides to the C-termini of the sfGFP and mCherry fluorescent proteins enables selective labeling of the ends or the sidewalls of single walled carbon nanotubes. By installing a gold-binding peptide or a single cysteine residue in carbon-binding variants of sfGFP, we further produce heterobifunctional solid-binding proteins that support the decoration of nanotubes sidewalls or termini with gold nanoparticles. The approach described here is generic and should prove useful for the controlled assembly of other hybrid materials.

Two-Channel Bioprotonic Photodetector.

Merging biological systems with electronic components requires converting biological ionic currents into electrical signals. Previously, we coupled green-light-activated transport of protons by a palladium-binding version of H. turkmenica deltarhodopsin (HtdR) with electronic signal generation by exploiting palladium hydride (PdHx) formation on palladium (Pd) electrodes. Here, we broaden the scope of these devices by showing that blue proteorhodopsin (BPR) from marine bacteria is a suitable proton pump for expanding their spectral range. After engineering BPR for Pd binding and high-level expression in E. coli and after demonstrating that the fused Pd-binding domain is properly oriented to bring exiting protons to the surface of Pd/PdHx contacts, we take advantage of the pH tunability of the BPR absorption spectrum to construct HtdR- and BPR-based devices with light absorption maxima, and thus photocurrent maxima, separated by 37 nm. These devices exhibit wavelength-dependent photocurrent production when illuminated between 450 and 600 nm, opening the door to the development of biological cameras.

Role of the signal sequence in proteorhodopsin biogenesis in E. coli.

Blue-absorbing proteorhodopsin (BPR) from marine bacteria is a retinal-bound, light-activated, outwards proton transporter containing seven α-helical transmembrane segments (TMS). It is synthesized as a precursor species (pre-BPR) with a predicted N-terminal signal sequence that is cleaved to yield the mature protein. While optimizing the production of BPR in Escherichia coli to facilitate the construction of bioprotonic devices, we observed significant pre-BPR accumulation in the inner membrane and explored signal sequence requirements and export pathway. We report here that BPR does not rely on the Sec pathway for inner membrane integration, and that although it greatly enhances yields, its signal sequence is not necessary to obtain a functional product. We further show that an unprocessable version of pre-BPR obtained by mutagenesis of the signal peptidase I site exhibits all functional attributes of the wild-type protein and has the advantage of being produced at higher levels. Our results are consistent with the BPR signal sequence being recognized by the signal recognition particle (SRP; a protein that orchestrates the cotranslational biogenesis of inner membrane proteins) and serving as a beneficial "pro" domain rather than a traditional secretory peptide.

A Self-Assembling Two-Dimensional Protein Array is a Versatile Platform for the Assembly of Multicomponent Nanostructures.

Rationally designed two-dimensional (2D) arrays that support the assembly of nanoscale components are of interest for catalysis, sensing, and biomedical applications. The computational redesign of a protein called TTM that undergoes calcium-induced self-assembly into nanostructured lattices capable of growing to dozens of micrometers are previously reported. The work demonstrates here that the N- and C-termini of the constituent monomers are solvent-accessible and that they can be modified with a hexahistidine extension, a gold-binding peptide, or a biotinylation tag to decorate nickel-nitriloacetic acid beads with self-assembled protein islands, conjugate gold nanoparticles to planar arrays, or control the immobilization density of avidin molecules onto 2D lattices through co-polymerization of biotinylated and wild type TTM monomers. These results showcase the potential of TTM as a versatile 2D scaffold for the fabrication of hierarchical structures comprising a broad range of nanoscale elements.

Affinity purification of Car9-tagged proteins on silica matrices: Optimization of a rapid and inexpensive protein purification technology.

Car9, a dodecapeptide identified by cell surface display for its ability to bind to the edge of carbonaceous materials, also binds to silica with high affinity. The interaction can be disrupted with l-lysine or l-arginine, enabling a broad range of technological applications. Previously, we reported that C-terminal Car9 extensions support efficient protein purification on underivatized silica. Here, we show that the Car9 tag is functional and TEV protease-excisable when fused to the N-termini of target proteins, and that it supports affinity purification under denaturing conditions, albeit with reduced yields. We further demonstrate that capture of Car9-tagged proteins is enhanced on small particle size silica gels with large pores, that the concomitant problem of nonspecific protein adsorption can be solved by lysing cells in the presence of 0.3% Tween 20, and that efficient elution is achieved at reduced l-lysine concentrations under alkaline conditions. An optimized small-scale purification kit incorporating the above features allows Car9-tagged proteins to be inexpensively recovered in minutes with better than 90% purity. The Car9 affinity purification technology should prove valuable for laboratory-scale applications requiring rapid access to milligram-quantities of proteins, and for preparative scale purification schemes where cost and productivity are important factors.

Streamlined Synthesis and Assembly of a Hybrid Sensing Architecture with Solid Binding Proteins and Click Chemistry.

Combining bioorthogonal chemistry with the use of proteins engineered with adhesive and morphogenetic solid-binding peptides is a promising route for synthesizing hybrid materials with the economy and efficiency of living systems. Using optical sensing of chloramphenicol as a proof of concept, we show here that a GFP variant engineered with zinc sulfide and silica-binding peptides on opposite sides of its ß-barrel supports the fabrication of protein-capped ZnS:Mn nanocrystals that exhibit the combined emission signatures of organic and inorganic fluorophores. Conjugation of a chloramphenicol-specific DNA aptamer to the protein shell through strain-promoted azide-alkyne cycloaddition and spontaneous concentration of the resulting nanostructures onto SiO2 particles mediated by the silica-binding sequence enables visual detection of environmentally and clinically relevant concentrations of chloramphenicol through analyte-mediated inner filtering of sub-330 nm excitation light.