Characterization of recombinant human lactoferrin expressed in Komagataella phaffii.

This work presents a thorough characterization of Helaina recombinant human lactoferrin (rhLF, Effera™) expressed in a yeast system at an industrial scale for the first time. Proteomic analysis confirmed that its amino acid sequence is identical to that of native human LF. N-linked glycans were detected at three known glycosylation sites, namely, Asparagines-156, -497, and -642 and they were predominantly oligomannose structures having five to nine mannoses. Helaina rhLF's protein secondary structure was nearly identical to that of human milk lactoferrin (hmLF), as revealed by microfluidic modulation spectroscopy. Results of small-angle X-ray scattering (SAXS) and analytical ultracentrifugation analyses confirmed that, like hmLF, Helaina rhLF displayed well-folded globular structures in solution. Reconstructed solvent envelopes of Helaina rhLF, obtained through the SAXS analysis, demonstrated a remarkable fit with the reported crystalline structure of iron-bound native hmLF. Differential scanning calorimetry investigations into the thermal stability of Helaina rhLF revealed two distinct denaturation temperatures at 68.7 ± 0.9 °C and 91.9 ± 0.5 °C, consistently mirroring denaturation temperatures observed for apo- and holo-hmLF. Overall, Helaina rhLF differed from hmLF in the N-glycans they possessed; nevertheless, the characterization results affirmed that Helaina rhLF was of high purity and exhibited globular structures closely akin to that of hmLF.

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.

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.

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.

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.

Self-Immobilization of Car9 Fusion Proteins within High Surface Area Silica Sol-Gels and Dynamic Control of Protein Release.

Protein entrapment within silica matrices during sol-gel formation is an effective way of producing biocatalysts with high load, activity retention, and minimal leaching. On the other hand, mesoporous silica materials have been favored for diffusional control of protein delivery because of their regular pore size and morphology and in spite of the drawback of requiring post-synthesis loading with cargo proteins. Here, we describe a hybrid technology in which fusion of the silica-binding Car9 dodecapeptide to model fluorescent proteins allows for their simultaneous entrapment and surface immobilization within sol-gel monoliths that can be fabricated in air and oil phases. Spherical particles produced by injecting a mixture of silicic acid and Car9-tagged proteins in silicone oil exhibit high surface area (>400 m2/g), 15-nm-diameter mean pore size and homogeneous protein loading. Incubation in arginine-containing buffer disrupts the interaction between Car9 extensions and silica surfaces and triggers the continuous or discontinuous (on/off) release of cargo proteins with pH-tunable kinetics. This simple approach for producing hybrid silica materials that stably encapsulate and release one or more Car9-tagged proteins in a single step may prove useful for applications requiring dynamic control of protein concentration.

PEG-labeled nucleotides and nanopore detection for single molecule DNA sequencing by synthesis.

We describe a novel single molecule nanopore-based sequencing by synthesis (Nano-SBS) strategy that can accurately distinguish four bases by detecting 4 different sized tags released from 5'-phosphate-modified nucleotides. The basic principle is as follows. As each nucleotide is incorporated into the growing DNA strand during the polymerase reaction, its tag is released and enters a nanopore in release order. This produces a unique ionic current blockade signature due to the tag's distinct chemical structure, thereby determining DNA sequence electronically at single molecule level with single base resolution. As proof of principle, we attached four different length PEG-coumarin tags to the terminal phosphate of 2'-deoxyguanosine-5'-tetraphosphate. We demonstrate efficient, accurate incorporation of the nucleotide analogs during the polymerase reaction, and excellent discrimination among the four tags based on nanopore ionic currents. This approach coupled with polymerase attached to the nanopores in an array format should yield a single-molecule electronic Nano-SBS platform.

Effect of surfactant mixtures on skin structure and barrier properties.

We investigated the effect of two commonly studied surfactants, sodium dodecyl sulfate (SDS) and dodecyl trimethylammonium bromide (C(12)TAB), on skin barrier properties. Using skin conductivity, FT-IR of stratum corneum samples, and penetration of radiolabelled SDS, we determined that addition of C(12)TAB lowers the ability of SDS to perturb skin's barrier properties. Ultrafiltration experiments revealed that addition of C(12)TAB serves to decrease the concentration of monomers and sub-micellar aggregates. None of the measured skin properties including enhancement of skin conductivity, perturbation of lipid structure and skin concentration of SDS correlated with the total SDS concentration in the donor compartment (i.e., the total SDS concentration). However, all these parameters correlated well against the concentration of monomers and sub-micellar aggregates. These findings provide the evidence of the importance of monomer and sub-micellar components in altering skin barrier properties.