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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.

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.

In this work we synthesize gold nanostructures using a liquid-handling robot and present new data analysis methods to quantitatively compare the effect of peptides and peptide modifications on gold nanoparticle synthesis outcomes. The peptides used were gold binding peptides Z2 and AG3, as well as five Z2 variants obtained by sequence modification or conjugation of a lipid tail. Four different concentrations of peptide, reducing agent (HEPES), and precursor (\ce{HAuCl4}) were used to synthesize 64 different reagent combinations for each of the seven peptides. Each sample was characterized using UV-Vis spectroscopy, which serves as a proxy for changes in nanoparticle structure, and enabled comparisons of how peptide modifications and reagent conditions affect synthesis outcomes. We then used functional data analysis to extract a pairwise signal correlation distance between each of the peptides. The signal correlation distance quantified how different the set of 64 spectra, and therefore the synthesis outcomes, was from one peptide to the other. We show that substitution of methionine for isoleucine in Z2 has a profound impact on synthesis outcomes when compared with conjugation of a lipid tail. Electron microscopy and ultra-small angle X-ray scattering were used to corroborate our conclusions from spectroscopy experiments by directly characterizing the structure of a smaller set of samples. Scanning electron microscopy revealed interconnected plate-like structures in samples prepared with a Z2 variant with all methionines substituted by isoleucine. Z2 peptides modified with a lipid tail on the other hand, formed nanoparticles which were more colloidally stable than those prepared with non-lipidated peptides, contained no plate-like particles, and whose size depended on the ratio of peptide to precursor.

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.

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.

Low-density aerogel materials are a promising platform for delivering lipophilic drugs with poor water solubility, but to date, the loading of aerogels with pharmaceuticals in crystalline form has not been reported other than by expensive supercritical loading techniques. Here, we demonstrate a simple and low-cost liquid-phase impregnation method to load the model drug coenzyme Q10 (CoQ10) within carbon aerogels. By controlling the chemical microstructure of the aerogel to exhibit π-conjugated (sp2) bonding via addition of graphene oxide, the rate of adsorption and total loading of CoQ10 is significantly enhanced. Furthermore, the adsorbed CoQ10 adopts an as-yet unreported crystalline phase that differs from the bulk material, not only offering the potential advantages of crystalline materials for drug delivery, but also a means to control the microstructure of this important lipophilic pharmaceutical.

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.

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.

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.

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.

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

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.

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 β-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 SiO(2) 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.

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.

Fusion of a palladium-binding peptide to an archaeal rhodopsin promotes intimate integration of the lipid-embedded membrane protein with a palladium hydride protonic contact. Devices fabricated with the palladium-binding deltarhodopsin enable light-activated conversion of protonic currents to electronic currents with on/off responses complete in seconds and a nearly tenfold increase in electrical signal relative to those made with the wild-type protein.

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.

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.

The formation of condensed matter typically involves a tradeoff between structural order and flexibility. As the extent and directionality of interactions between atomic or molecular components increase, materials generally become more ordered but less compliant, and vice versa. Nevertheless, high levels of structural order and flexibility are not necessarily mutually exclusive; there are many biological (such as microtubules1,2, flagella3, viruses4,5) and synthetic assemblies (for example, dynamic molecular crystals6–9 and frameworks10–13) that can undergo considerable structural transformations without losing their crystalline order and that have remarkable mechanical properties8,14,15 that are useful in diverse applications, such as selective sorption16, separation17, sensing18 and mechanoactuation19. However, the extent of structural changes and the elasticity of such flexible crystals are constrained by the necessity to maintain a continuous network of bonding interactions between the constituents of the lattice. Consequently, even the most dynamic porous materials tend to be brittle and isolated as microcrystalline powders14, whereas flexible organic or inorganic molecular crystals cannot expand without fracturing. Owing to their rigidity, crystalline materials rarely display self-healing behaviour20. Here we report that macromolecular ferritin crystals with integrated hydrogel polymers can isotropically expand to 180 per cent of their original dimensions and more than 500 per cent of their original volume while retaining periodic order and faceted Wulff morphologies. Even after the separation of neighbouring ferritin molecules by 50 ångströms upon lattice expansion, specific molecular contacts between them can be reformed upon lattice contraction, resulting in the recovery of atomic-level periodicity and the highest-resolution ferritin structure reported so far. Dynamic bonding interactions between the hydrogel network and the ferritin molecules endow the crystals with the ability to resist fragmentation and self-heal efficiently, whereas the chemical tailorability of the ferritin molecules enables the creation of chemically and mechanically differentiated domains within single crystals.

We have combined fusion of oligomers with cyclic symmetry and alanine substitutions to eliminate clashes and produce proteins that self-assemble into 2-D arrays upon addition of calcium ions. Using TEM, AFM, small-angle X-ray scattering, and fluorescence microscopy, we show that the designed lattices which are 5 nm high with p3 space group symmetry and 7.25 nm periodicity self-assemble into structures that can exceed 100 μm in characteristic length. The versatile strategy, experimental approach, and hexagonal arrays described herein should prove valuable for the engineering of functional nanostructured materials in 2-D.

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.

We describe a new affinity purification tag called Car9 that confers proteins to which it is fused micromolar affinity for unmodified silica. When appended to the C-terminus of GFPmut2 through a flexible linker, Car9 promotes efficient adsorption to silica gel and the fusion protein can be released from the particles by incubation with L-lysine. Using a silica gel column and the lysine elution approach in fast protein liquid chromatography (FPLC) mode, Car9-tagged versions of GFPmut2, mCherry and maltose binding protein (MBP) can be recovered from clarified lysates with a purity of 80-90%. Capitalizing on silica's ability to handle large pressure drops, we further show that it is possible to go from cell lysates to purified protein in less than 15 min using a fully disposable device. Finally, we demonstrate that the linker-Car9 region is susceptible to proteolysis by E. coli OmpT and take advantage of this observation to excise the C-terminal extension of GFPmut2-Car9 by incubating purified fusion protein with cells that overproduce the outer membrane protease OmpT. The set of strategies described herein, should reduce the cost of affinity purification by at least 10-fold, cut down purification times to minutes, and allow for the production of proteins with native (or nearly native) termini from their C-terminally-tagged versions.

An absorbent gel has been integrated into the void space of a protein crystal to yield a remarkable self-healing material — it recovers its molecular order after several cycles of expansion and contraction. An absorbent gel has been integrated into the void space of a protein crystal to yield a remarkable self-healing material — it recovers its molecular order after several cycles of expansion and contraction.

Fusion of disulfide-constrained or linear versions of the Car9 dodecapeptide to model fluorescent proteins support their on-contact and oriented immobilization onto unmodified glass. Bound proteins can be released and the surface regenerated by incubation with L-lysine. This noncovalent chemistry enables rapid and reversibe microcontact printing of tagged proteins and speeds up the production of bicontinuous protein patterns.

Silver toxicity is a problem that microorganisms face in medical and environmental settings. Through exposure to silver compounds, some bacteria have adapted to growth in high concentrations of silver ions. Such adapted microbes may be dangerous as pathogens but, alternatively, could be potentially useful in nanomaterial-manufacturing applications. While naturally adapted isolates typically utilize efflux pumps to achieve metal resistance, we have engineered a silver-tolerant Escherichia coli strain by the use of a simple silver-binding peptide motif. A silver-binding peptide, AgBP2, was identified from a combinatorial display library and fused to the C terminus of the E. coli maltose-binding protein (MBP) to yield a silver-binding protein exhibiting nanomolar affinity for the metal. Growth experiments performed in the presence of silver nitrate showed that cells secreting MBP-AgBP2 into the periplasm exhibited silver tolerance in a batch culture, while those expressing a cytoplasmic version of the fusion protein or MBP alone did not. Transmission electron microscopy analysis of silver-tolerant cells revealed the presence of electron-dense silver nanoparticles. This is the first report of a specifically engineered metal-binding peptide exhibiting a strong in vivo phenotype, pointing toward a novel ability to manipulate bacterial interactions with heavy metals by the use of short and simple peptide motifs. Engineered metal-ion-tolerant microorganisms such as this E. coli strain could potentially be used in applications ranging from remediation to interrogation of biomolecule-metal interactions in vivo .

Designer proteins that incorporate solid-binding peptides hold promise to control the nucleation, growth, morphology, and assembly of inorganic phases under mild conditions of temperature and pressure. However, protein-aided nanofabrication remains more art than science and some materials can only be synthesized at temperatures that cause most mesophilic proteins to unfold. Using zinc oxide (ZnO) synthesis at 70°C as case study, we show here that seemingly unimportant variables, such as the carry-over concentration of Tris buffer and the “empty” host protein scaffold can exert a significant influence on materials morphology. We also show that, once well-controlled conditions are established, thermodynamic predictions and adsorption isotherms are powerful tools to understand how various ZnO-binding sequence inserted within the thermostable framework of Escherichia coli thioredoxin A (TrxA) affect inorganic morphogenesis. Biotechnol. Bioeng. 2012; 109:1912–1918. © 2012 Wiley Periodicals, Inc.

A minimized protein consisting of a linear ZnS-binding peptide fused to an antibody-binding domain supports the one-step aqueous synthesis of Mn-doped ZnS nanocrystals that exhibit smaller size, brighter fluorescence and improved antibody-binding relative to those made with the original designer protein.

Because membrane proteins are difficult to express, our understanding of their structure and function is lagging. In Escherichia coli, α-helical membrane protein biogenesis usually involves binding of a nascent transmembrane segment (TMS) by the signal recognition particle (SRP), delivery of the SRP-ribosome nascent chain complexes (RNC) to FtsY, a protein that serves as SRP receptor and docks to the SecYEG translocon, cotranslational insertion of the growing chain into the translocon, and lateral transfer, packing and folding of TMS in the lipid bilayer in a process that may involve chaperone YidC. Here, we explored the feasibility of reprogramming this pathway to improve the production of recombinant membrane proteins in exponentially growing E. coli with a focus on: (i) eliminating competition between SRP and chaperone trigger factor (TF) at the ribosome through gene deletion; (ii) improving RNC delivery to the inner membrane via SRP overexpression; and (iii) promoting substrate insertion and folding in the lipid bilayer by increasing YidC levels. Using a bitopic histidine kinase and two heptahelical rhodopsins as model systems, we show that the use of TF-deficient cells improves the yields of membrane-integrated material threefold to sevenfold relative to the wild type, and that whereas YidC coexpression is beneficial to the production of polytopic proteins, higher levels of SRP have the opposite effect. The implications of our results on the interplay of TF, SRP, YidC, and SecYEG in membrane protein biogenesis are discussed.

The signal recognition particle (SRP) dependent secretion pathway is as an attractive alternative to Sec-dependent export for the production of disulfide-bonded and/or fast-folding recombinant proteins in the Escherichia coli periplasm. SRP, which shares a ribosomal attachment site with the molecular chaperone trigger factor (TF), recognizes highly hydrophobic signal sequence as they emerge from the ribosome and delivers ribosome nascent chain complexes to FtsY for subsequent cotranslational translocation of target proteins across the SecYEG pore. However, like in the case of Sec-dependent export, secretory yields can be limited by the accumulation of precursor proteins in the cytoplasm. Using leech carboxypeptidase inhibitor (LCI) fused to the SRP-dependent DsbA signal sequence as a model system, we show that a null mutation in the gene encoding TF (Deltatig) or SRP co-expression reduce pre-LCI accumulation by half, and that quantitative export can be achieved by combining the two strategies. Interestingly, enhanced precursor processing did not alter periplasmic LCI levels but increased the amount of protein excreted in the growth medium. All mature LCI was nearly fully active and an 80% increase in productivity was achieved in Deltatig cells alone due to their faster growth. Our results show that competition between SRP and TF can interfere with efficient export of recombinant proteins targeted to the SRP pathway and establish TF-deficient strains and SRP co-expression as a simple solution to improve yields.

Quantum dots (QDs) are a powerful alternative to organic dyes and fluorescent proteins for biological and biomedical applications. These semiconductor nanocrystals are traditionally synthesized above 200 degrees C in organic solvents using toxic and costly precursors, and further steps are required to conjugate them to a biological ligand. Here, we describe a simple, aqueous route for the one-pot synthesis of antibody-derivatized zinc sulfide (ZnS) immuno-QDs. In this strategy, easily expressed and purified fusion proteins perform the dual function of nanocrystal mineralizers through ZnS binding sequences identified by cell surface display and adaptors for immunoglobin G (IgG) conjugation through a tandem repeat of the B domain of Staphylococcus aureus protein A. Although approximately 4.3 nm ZnS wurtzite cores could be biomineralized from either zinc chloride or zinc acetate precursors, only the latter salt gives rise to protein-coated QDs with long shelf life and narrow hydrodynamic diameters (8.8 +/- 1.4 nm). The biofabricated QDs have a quantum yield of 2.5% and blue-green ensemble emission with contributions from the band-edge at 340 nm and from trap states at 460 and 665 nm that are influenced by the identity of the protein shell. Murine IgG(1) antibodies exhibit high affinity (K(d) = 60 nM) for the protein shell, and stable immuno-QDs with a hydrodynamic diameter of 14.1 +/- 1.3 nm are readily obtained by mixing biofabricated nanocrystals with human IgG.

Here, we exploit the ability of electrodeposition to decouple materials nucleation and growth to investigate how engineered biomineralizing proteins control inorganic morphogenesis. Using a silver-binding designer protein at concentrations proximal to its equilibrium dissociation constant, we produce rosettelike silver particles instead of the pentagonal twinned crystals obtained without additive or with a wild-type protein that nonspecifically adsorbs to silver. We correlate the difference in silver growth habit to specific interactions between the designer protein and the {111} faces of polycrystalline silver nuclei. Our results suggest that electrodeposition is a powerful tool to interrogate protein−inorganic interactions and to screen solid-binding proteins for shape-changing activity.

Hsp31 is a stress-inducible molecular chaperone involved in the management of protein misfolding at high temperatures and in the development of acid resistance in starved E. coli. Each subunit of the Hsp31 homodimer consists of two structural domains connected by a flexible linker that sits atop a continuous tract of nonpolar residues adjacent to a hydrophobic bowl defined by the dimerization interface. Previously, we proposed that while the bowl serves as a binding site for partially folded species at physiological temperatures, chaperone function under heat shock conditions requires that folding intermediates further anneal to high-affinity binding sites that become uncovered upon thermally induced motion of the linker. In support of a mechanism requiring that client proteins first bind to the bowl, we show here that fusion of a 20-residue-long hexahistidine tag to the N-termini of Hsp31 abolishes chaperone activity at all temperatures by inducing reversible structural changes that interfere with substrate binding. We further demonstrate that extending the C-termini of Hsp31 with short His tags selectively suppresses chaperone function at high temperatures by interfering with linker movement. The structural and functional sensitivity of Hsp31 to lengthening is consistent with the high degree of conservation of class I Hsp31 orthologs and will serve as a cautionary tale on the implications of affinity tagging.

We have used electrochemical processing to fabricate ordered arrays of metals and metal oxides on surfaces at densities exceeding 10 12 cm -2 , on mm 2 areas, and with typical feature sizes of 2-3 nm. This is achieved via masks obtained from naturally occurring proteins that assemble into two-dimensional crystals containing internal porous structure within each unit cell of the crystalline lattice. We have proven this process with bacterial cell surface proteins (S-layer proteins) from Deinococcus radiodurans and Sporosarcina ureae. Each of these S-layer proteins has unique lattice geometry and internal structure. Substrates are coated by adsorption from a dilute suspension of purified, stabilized protein extract. Electrochemical deposition proceeds through solvent accessible pores of the S-layer crystal to build surface structures with nanometer scale feature sizes and spacings precisely matching the geometry of the protein "mask". Comparisons between the structure of the electrodeposited material through the protein mask and the protein surface topography suggest that the S-layers of D. radiodurans possess pores providing straight through-holes to the surface, whereas the S-layers of S. ureae presents a more tortuous pathway to the electrode surface.

A high-yield, easy to master method for preparing electron transparent metal, oxide, and carbon ultrathin film substrates suitable for direct nano/micro-fabrication and transmission electron microscopy (TEM) is presented. To demonstrate the versatility of these substrates for fabrication processes, we use e-beam lithography, self-assembled colloidal and protein templates, and microcontact printing to create patterned masks for subsequent electrodeposition of two dimensional and three dimensional structures. The electrodeposited structures range in scale from a few nanometers to a few micrometers in characteristic dimensions. Because fabrication occurs directly on ultrathin films, TEM analysis of the resulting materials and buried interfaces is straightforward without any destructive sample preparation. We show that all the normal TEM analytical methods (imaging, diffraction, electron and X-ray spectroscopies) are compatible with the fabricated structures and the thin film substrates. These electron transparent substrates have largely rendered the need for TEM sample preparation on fabricated structures obsolete in our lab.

In biological systems, intercellular communication is mediated by membrane proteins and ion channels that regulate traffic of ions and small molecules across cell membranes. A bioelectronic device with ion channels that control ionic flow across a supported lipid bilayer (SLB) should therefore be ideal for interfacing with biological systems. Here, we demonstrate a biotic–abiotic bioprotonic device with Pd contacts that regulates proton (H+) flow across an SLB incorporating the ion channels Gramicidin A (gA) and Alamethicin (ALM). We model the device characteristics using the Goldman–Hodgkin–Katz (GHK) solution to the Nernst–Planck equation for transport across the membrane. We derive the permeability for an SLB integrating gA and ALM and demonstrate pH control as a function of applied voltage and membrane permeability. This work opens the door to integrating more complex H+ channels at the Pd contact interface to produce responsive biotic–abiotic devices with increased functionality., Conventional electronics use electrons as charge carriers whereas biological systems use ions, confounding integration of electronics with biology. Here the authors show voltage-regulated flow of protons across a supported lipid bilayer using the ion channel Gramicidin A and the voltage-gated ion channel Alamethicin.

For nearly 40 years, therapeutic proteins have been stabilized by chemical conjugation of polyethylene glycol (PEG), but recently zwitterionic materials have proved to be a more effective substitute. In this work, we demonstrate that genetic fusion of alternating-charge extensions consisting of anionic glutamic acid (E) and cationic lysine (K) is an effective strategy for protein stabilization. This bioinspired "EKylation" method not only confers the stabilizing benefits of poly(zwitterions) but also allows for rapid biosynthesis of target constructs. Poly(EK) peptides of different predetermined lengths were appended to the C-terminus of a native β-lactamase and its destabilized TEM-19 mutant. The EK-modified enzymes retained biological activity and exhibited increased stability to environmental stressors such as high temperature and high-salt solutions. This one-step strategy provides a broadly applicable alternative to synthetic polymer conjugation that is biocompatible and degradable.

Error-prone PCR was used to isolate Photinus pyralis luciferase mutants producing bright light in the red-orange region of the spectrum. All mutations were clustered in the beta5-alpha10-beta6 region of N-terminal subdomain B and appear to affect bioluminescence color by modulating the position of the Ser314-Leu319 mobile loop with respect to the putative active site. Two red variants (Q283R and S284G) and one orange mutant (S293P) contained a single substitution. Although the remaining orange variant contained two mutations, L287I mainly contributed to the color change. Emission spectra collected on whole cells at pH 7.0 revealed that while a single peak of lambdamax approximately 605 nm accounts for red light production by the Q283R and S284G variants, orange light results from the contribution of two peaks of lambdamax approximately 560 and 600 nm. All spectra underwent a red-shift when cells were assayed under acidic conditions, whereas a blue-shift was observed at pH 8.0, indicating that the internal pH of Escherichia coli is close to the external pH shortly after imposition of acid or alkaline stress. In addition, changes in assay pH led to bimodal emission spectra, lending support to the idea that bioluminescence color is determined by the relative contribution of yellow-green and red-orange peaks. The set of multicolored luciferase mutants described here may prove useful for a variety of applications including biosensing, pH monitoring, and tissue and animal imaging.

Eubacteria synthesize a full-length (ClpB95) and a N-terminally truncated (ClpB80) version of the ClpB disaggregase owing to the presence of a translation initiation site within the clpB transcript. Why these two isoforms have been evolutionary conserved is poorly understood. Here, we constructed a series of E. coli strains and plasmids allowing production of the ClpB95/ClpB80 pair, ClpB95 alone, or ClpB80 alone from near physiological concentrations to a 6–10-fold excess over normal cellular levels. We found that although overexpressed ClpB95 or ClpB80 can independently restore basal thermotolerance to Δ clpB cells, strains expressing ClpB80 from the clpB chromosomal locus do not exhibit increased resistance to thermal killing at 50 °C relative to clpB null cells. Furthermore, synthesis of physiological levels of ClpB95 is less effective than coordinated expression of ClpB95/ClpB80 in protecting E. coli from thermal killing. These results provide an explanation for the conservation of the two ClpB isoforms in eubacteria and are consistent with the fact that wild type E. coli maintains the ClpB80 to ClpB95 ratio at a nearly constant value of 0.4–0.5 under a variety of stress conditions.

▪ Abstract Molecular biomimetics can be defined as mimicking function, synthesis, or structure of materials and systems at the molecular scale using biological pathways. Here, inorganic-binding polypeptides are used as molecular building blocks to control assembly and formation of functional inorganic and hybrid materials and systems for nano- and nanobiotechnology applications. These polypeptides are selected via phage or cell surface display technologies and modified by molecular biology to tailor their binding and multifunctionality properties. The potential of this approach in creating new materials systems with useful physical and biological properties is enormous. This mostly stems from molecular recognition and self-assembly characteristics of the polypeptides plus the added advantage of genetic manipulation of their composition and structure. In this review, we highlight the basic premises of molecular biomimetics, describe the approaches in selecting and engineering inorganic-binding polypeptides, and present examples of their utility as molecular linkers in current and future applications.

Escherichia coli Hsp31 is a homodimeric protein that exhibits chaperone activity in vitro and is a representative member of a recently recognized family of heat shock proteins (Hsps). To gain insights on Hsp31 cellular function, we deleted the hchA gene from the MC4100 chromosome and combined the resulting null allele with lesions in other cytoplasmic chaperones. Although the hchA mutant only exhibited growth defects when cultivated at 48 degrees C, loss of Hsp31 had a strong deleterious effect on the ability of cells to survive and recover from transient exposure to 50 degrees C, and led to the enhanced aggregation of a subset of host proteins at this temperature. The absence of Hsp31 did not significantly affect the ability of the ClpB-DnaK-DnaJ-GrpE system to clear thermally aggregated proteins at 30 degrees C suggesting that Hsp31 does not possess disaggregase activity. Although it had no effect on the growth of groES30, Delta clpB or Delta ibpAB cells at high temperatures, the hchA deletion aggravated the temperature sensitive phenotype of dnaK756 and grpE280 mutants and led to increased aggregation in stressed dnaK756 cells. On the basis of biochemical, structural and genetic data, we propose that Hsp31 acts as a modified holding chaperone that captures early unfolding intermediates under prolonged conditions of severe stress and releases them when cells return to physiological conditions. This additional line of defence would complement the roles of DnaK-DnaJ-GrpE, ClpB and IbpB in the management of thermally induced cellular protein misfolding.

Proteins, through their unique and specific interactions with other macromolecules and inorganics, control structures and functions of all biological hard and soft tissues in organisms. Molecular biomimetics is an emerging field in which hybrid technologies are developed by using the tools of molecular biology and nanotechnology. Taking lessons from biology, polypeptides can now be genetically engineered to specifically bind to selected inorganic compounds for applications in nano- and biotechnology. This review discusses combinatorial biological protocols, that is, bacterial cell surface and phage-display technologies, in the selection of short sequences that have affinity to (noble) metals, semiconducting oxides and other technological compounds. These genetically engineered proteins for inorganics (GEPIs) can be used in the assembly of functional nanostructures. Based on the three fundamental principles of molecular recognition, self-assembly and DNA manipulation, we highlight successful uses of GEPI in nanotechnology.

Heat shock proteins (Hsps) play essential protective roles under stress conditions by preventing the formation of protein aggregates and degrading misfolded proteins. EcHsp31, the yedU ( hchA ) gene product, is a representative member of a family of chaperones that alleviates protein misfolding by interacting with early unfolding intermediates. The 1.6-Å crystal structure of the EcHsp31 dimer reveals a system of hydrophobic patches, canyons, and grooves, which may stabilize partially unfolded substrate. The presence of a well conserved, yet buried, triad in each two-domain subunit suggests a still unproven hydrolytic function of the protein. A flexible extended linker between the A and P domains may play a role in conformational flexibility and substrate binding. The α-β sandwich of the EcHsp31 monomer shows structural similarity to PhPI, a protease belonging to the DJ-1 superfamily. The structure-guided sequence alignment indicates that Hsp31 homologs can be divided in three classes based on variations in the P domain that dramatically affect both oligomerization and catalytic triad formation.

To gain insights into the in vivo folding and assembly of bacterial chaperonins, groEL was subjected to insertion mutagenesis using transposon IS lacZ /in. Four GroEL-LacZ fusions and the corresponding insertion mutants were obtained after residues 34, 90, 291, and 367. Apical domain insertion mutants GroEL291 and GroEL367 were degraded into monomeric 30- and 40-kDa fragments, respectively. Only the latter was fully soluble, suggesting that proper isomerization of an essentially complete apical domain is required for efficient protomer folding. Truncated variants were inactive as minichaperones as they failed to restore the growth of groEL140 cells at 43 °C whether or not GroES was co-expressed. A 31-residue insertion in equatorial helix D led to complete degradation of GroEL90. By contrast, extraneous amino acids were tolerated at equatorial position 34, indicating that this region is highly flexible. Nevertheless, GroEL34 did not fold as efficiently as authentic GroEL and reached only a heptameric conformation.

The Escherichia coli chromosome contains several uncharacterized heat-inducible loci that may encode novel molecular chaperones or proteases. Here we show that the 31-kDa product of the yedU gene is an efficient homodimeric molecular chaperone that is conserved in a number of pathogenic eubacteria and fungi. Heat shock protein (Hsp) 31 relies on temperature-driven conformational changes to expose structured hydrophobic domains that are likely responsible for substrate binding. Complementing the function of refolding, remodeling, and holding chaperones, Hsp 31 preferentially interacts with early unfolding intermediates and rapidly releases them in an active form after transfer to low temperatures. Although Hsp 31 does not appear to exhibit intrinsic ATPase activity, binding of ATP at high temperatures restricts the size or availability of the substrate binding site, thereby modulating chaperone activity. The possible role of ATP in coordinating the function of the cellular complement of molecular chaperones is discussed.

DnaK-DnaJ-GrpE and GroEL-GroES are the best-characterized molecular chaperone systems in the cytoplasm of Escherichia coli. A number of additional proteins, including ClpA, ClpB, HtpG and IbpA/B, act as molecular chaperones in vitro, but their function in cellular protein folding remains unclear. Here, we examine how these chaperones influence the folding of newly synthesized recombinant proteins under heat-shock conditions. We show that the absence of either CIpB or HtpG at 42 degrees C leads to increased aggregation of preS2-beta-galactosidase, a fusion protein whose folding depends on DnaK-DnaJ-GrpE, but not GroEL-GroES. However, only the deltaclpB mutation is deleterious to the folding of homodimeric Rubisco and cMBP, two proteins requiring the GroEL-GroES chaperonins to reach a proper conformation. Null mutations in clpA or the ibpAB operon do not affect the folding of these model substrates. Overexpression of ClpB, HtpG, IbpA/B or ClpA does not suppress inclusion body formation by the aggregation-prone protein preS2-S'-beta-galactosidase in wild-type cells or alleviate recombinant protein misfolding in dnaJ259, grpE280 or groES30 mutants. By contrast, higher levels of DnaK-DnaJ, but not GroEL-GroES, restore efficient folding in deltaclpB cells. These results indicate that ClpB, and to a lesser extent HtpG, participate in de novo protein folding in mildly stressed E. coli cells, presumably by expanding the ability of the DnaK-DnaJ-GrpE team to interact with newly synthesized polypeptides.

The ubiquitous hexahistidine purification tag has been used to conjugate proteins to the shell of CdSe:ZnS quantum dots (QDs) due to its affinity for surface-exposed Zn2+ ions but little attention has been paid to the potential of His-tagged proteins for mineralizing luminescent ZnS nanocrystals. Here, we compare the ability of free histidine, a His tag peptide, His-tagged thioredoxin (TrxA, a monomeric protein), and N- and C-terminally His-tagged versions of Hsp31 (a homodimeric protein) to support the synthesis of Mn-doped ZnS nanocrystals from aqueous precursors under mild conditions of pH (8.2) and temperature (37 °C). We find that: (1) it is possible to produce poor quality QDs when histidine is used at high (8 mM) concentration; (2) an increase in local histidine concentration through repetition of the amino acid as a His tag decreases the amount of needed reagent ≈10-fold and improves optical properties; (3) fusion of the same His tag to TrxA allows for ZnS:Mn QDs mineralization at micromolar concentrations; and (4) doubling the local hexahistidine concentration by exploiting Hsp31 dimerization further improves nanocrystal luminescence with the brightest particles obtained when His tags are spatially co-localized at the Hsp31 N-termini. Although hexahistidine tracts are not as efficient as combinatorially selected ZnS binding peptides at QD synthesis, it should be possible to use the large number of available His-tagged proteins and the synthesis approach described herein to produce luminescent nanoparticles whose protein shell carries a broad range of functions.

The small size and heavy metal composition of quantum dots (QDs) combined with their growing consumer product and biomedical research applications have generated concern over their safety. In an occupational setting where QD-enabled products are being manufactured, inhalation is a likely route of exposure. Since current research indicates that QDs could cause inflammation and toxicity in the respiratory tract, it is important that a variety of methods be available to further characterize this potential respiratory hazard. This chapter focuses primarily on in vivo methods for modeling the inhalation and assessing the pulmonary toxicity of QDs.

TolAI--II--beta-lactamase, a fusion protein consisting of the inner membrane and transperiplasmic domains of TolA followed by TEM--beta-lactamase associated with the inner membrane but remained confined to the cytoplasm when expressed at high level in Escherichia coli. Although the fusion protein was resistant to proteolysis in vivo, it was hydrolyzed during preparative SDS-polyacrylamide electrophoresis and when insoluble cellular fractions unfolded with 5 M urea were subjected to microdialysis. Inhibitor profiling studies revealed that both a metallo- and serine protease were involved in TolAI--II--beta-lactamase degradation under denaturing conditions. The in vitro degradation rates of the fusion protein were not affected when insoluble fractions were harvested from a strain lacking protease IV, but were significantly reduced when microdialysis experiments were conducted with material isolated from an isogenic ftsH1 mutant. Adenine nucleotides were not required for degradation, and ATP supplementation did not accelerate the apparent rate of TolAI--II--beta-lactamase hydrolysis under denaturing conditions. Our results indicate that the metalloprotease active site of FtsH remains functional in the presence of 3--5 M urea and suggest that the ATPase and proteolytic activities of FtsH can be uncoupled if the substrate is sufficiently unstructured. Thus, a key role of the FtsH AAA module appears to be the net unfolding of bound substrates so that they can be efficiently engaged by the protease active site.

Single-copy gene fusions between the lacZ reporter gene and Escherichia coli strains containing promoters induced by cold shock ( cspA ), cytoplasmic stress ( ibp ), or protein misfolding in the cell envelope (P3 rpoH ) were constructed and tested to determine their ability to detect antibacterial agents while simultaneously providing information on their cellular targets. Antibiotics that affect prokaryotic ribosomes selectively induced the cspA :: lacZ or ibp :: lacZ gene fusion, depending on their mode of action. The membrane-damaging peptide polymyxin B induced both the P3 rpoH :: lacZ and ibp :: lacZ fusions, while the β-lactam antibacterial agent carbenicillin activated only the P3 rpoH promoter. Nalidixic acid, a compound that causes DNA damage, downregulated β-galactosidase synthesis from P3 rpoH but had little effect on expression of the reporter enzyme from either the cspA or ibp promoter. All model antibiotics could be identified over a wide range of sublethal concentrations with signal-to-noise ratios between 2 and 11. A blue halo assay was developed to rapidly characterize the modes of action of antibacterial agents by visual inspection, and this assay was used to detect chloramphenicol secreted into the growth medium of Streptomyces venezuelae cultures. This simple system holds promise for screening natural or combinatorial libraries of antimicrobial compounds.