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Silk fibroin is a fiber-forming protein derived from the thread of Bombyx mori silkworm cocoons. This biocompatible protein, under the kosmotropic influence of potassium phosphate, can undergo supramolecular self-assembly driven by a random coil to β-sheet secondary structure transition. By leveraging concurrent nonspecific adsorption and self-assembly of silk fibroin, we demonstrate an interfacial phenomenon that yields adherent, defect-free nanothin protein coatings that grow continuously in time, without observable saturation in mass deposition. This noncovalent growth of silk fibroin coatings is a departure from traditionally studied protein adsorption phenomena, which generally yield adsorbed layers that saturate in mass with time and often do not completely cover the surface. Here, we explore the fundamental mechanisms of coating growth by examining the effects of coating solution parameters that promote or inhibit silk fibroin self-assembly. Results show a strong dependence of coating kinetics and structure on solution pH, salt species, and salt concentration. Moreover, coating growth was observed to occur in two stages: an early stage driven by protein-surface interactions and a late stage driven by protein-protein interactions. To describe this phenomenon, we developed a kinetic adsorption model with Langmuir-like behavior at early times and a constant steady-state growth rate at later times. Structural analysis by FTIR and photoinduced force microscopy show that small β-sheet-rich structures serve as anchoring sites for absorbing protein nanoaggregates, which is critical for coating formation. Additionally, β-sheets are preferentially located at the interface between protein nanoaggregates in the coating, suggesting their role in forming stable, robust coatings.

Two-dimensional (2D) materials provide a great opportunity for fabricating ideal membranes with ultrathin thickness for high-throughput separation. Graphene oxide (GO), owing to its hydrophilicity and functionality, has been extensively studied for membrane applications. However, fabrication of single-layered GO-based membranes utilizing structural defects for molecular permeation is still a great challenge. Optimization of the deposition methodology of GO flakes could offer a potential solution for fabricating desired nominal single-layered (NSL) membranes that can offer a dominant and controllable flow through structural defects of GO. In this study, a sequential coating methodology was adopted for depositing a NSL GO membrane, which is expected to have no or minimum stacking of GO flakes and thus ensure GO's structural defects as the major transport pathway. We have demonstrated effective rejection of different model proteins (bovine serum albumin (BSA), lysozyme, and immunoglobulin G (IgG)) by tuning the structural defect size via oxygen plasma etching. By generating appropriate structural defects, similar-sized proteins (myoglobin and lysozyme; molecular weight ratio (MWR): ∼1.14) were effectively separated with a separation factor of ∼6 and purity of 92%. These findings may provide new opportunities of using GO flakes for fabricating NSL membranes with tunable pores for applications in the biotechnology industry.

N95 respirator face masks serve as effective physical barriers against airborne virus transmission, especially in a hospital setting. However, conventional filtration materials, such as nonwoven polypropylene fibers, have no inherent virucidal activity, and thus, the risk of surface contamination increases with wear time. The ability of face masks to protect against infection can be likely improved by incorporating components that deactivate viruses on contact. We present a facile method for covalently attaching antiviral quaternary ammonium polymers to the fiber surfaces of nonwoven polypropylene fabrics that are commonly used as filtration materials in N95 respirators via ultraviolet (UV)-initiated grafting of biocidal agents. Here, C 12 -quaternized benzophenone is simultaneously polymerized and grafted onto melt-blown or spunbond polypropylene fabric using 254 nm UV light. This grafting method generated ultrathin polymer coatings which imparted a permanent cationic charge without grossly changing fiber morphology or air resistance across the filter. For melt-blown polypropylene, which comprises the active filtration layer of N95 respirator masks, filtration efficiency was negatively impacted from 72.5 to 51.3% for uncoated and coated single-ply samples, respectively. Similarly, directly applying the antiviral polymer to full N95 masks decreased the filtration efficiency from 90.4 to 79.8%. This effect was due to the exposure of melt-blown polypropylene to organic solvents used in the coating process. However, N95-level filtration efficiency could be achieved by wearing coated spunbond polypropylene over an N95 mask or by fabricating N95 masks with coated spunbond as the exterior layer. Coated materials demonstrated broad-spectrum antimicrobial activity against several lipid-enveloped viruses, as well as Staphylococcus aureus and Escherichia coli bacteria. For example, a 4.3-log reduction in infectious MHV-A59 virus and a 3.3-log reduction in infectious SuHV-1 virus after contact with coated filters were observed, although the level of viral deactivation varied significantly depending on the virus strain and protocol for assaying infectivity.

CRISPR-based detection of target DNA or RNA exploits a dual function, including target sequence-specific recognition followed by trans -cleavage activity of a collateral ssDNA linker between a fluorophore (F) and a quencher (Q), which amplifies a fluorescent signal upon cleavage. In this work, we have extended such dual functionality in a modified immunoassay format to detect a target protein, CXCL9, which is markedly elevated in the urine of kidney transplant recipients undergoing acute rejection episodes. To establish the "immuno-CRISPR" assay, we used anti-CXCL9 antibody-DNA barcode conjugates to target CXCL9 and amplify fluorescent signals via Cas12a-based trans -cleavage activity of FQ reporter substrates, respectively, and in the absence of an isothermal amplification step. To enhance detection sensitivity, the DNA barcode system was engineered by introducing multiple Cas12a recognition sites. Use of biotinylated DNA barcodes enabled self-assembly onto streptavidin (SA) to generate SA-DNA barcode complexes to increase the number and density of Cas12a recognition sites attached to biotinylated anti-CXCL9 antibody. As a result, we improved the rate of CXCL9 detection approximately 8-fold when compared to the use of a monomeric DNA barcode. The limit of detection (LOD) for CXCL9 using the immuno-CRISPR assay was 14 pg/mL, which represented an ∼7-fold improvement when compared to traditional HRP-based ELISA. Selectivity was shown with a lack of crossover reactivity with the related chemokine CXCL1. Finally, we successfully evaluated the presence of CXCL9 in urine samples from 11 kidney transplant recipients using the immuno-CRISPR assay, resulting in 100% accuracy to clinical CXCL9 determination and paving the way for use as a point-of-care noninvasive biomarker for the detection of kidney transplant rejection.

Piscidins are histidine-enriched antimicrobial peptides that interact with lipid bilayers as amphipathic α-helices. Their activity at acidic and basic pH in vivo makes them promising templates for biomedical applications. This study focuses on p1 and p3, both 22-residue-long piscidins with 68% sequence identity. They share three histidines (H3, H4, and H11), but p1, which is significantly more permeabilizing, has a fourth histidine (H17). This study investigates how variations in amphipathic character associated with histidines affect the permeabilization properties of p1 and p3. First, we show that the permeabilization ability of p3, but not p1, is strongly inhibited at pH 6.0 when the conserved histidines are partially charged and H17 is predominantly neutral. Second, our neutron diffraction measurements performed at low water content and neutral pH indicate that the average conformation of p1 is highly tilted, with its C-terminus extending into the opposite leaflet. In contrast, p3 is surface bound with its N-terminal end tilted toward the bilayer interior. The deeper membrane insertion of p1 correlates with its behavior at full hydration: an enhanced ability to tilt, bury its histidines and C-terminus, induce membrane thinning and defects, and alter membrane conductance and viscoelastic properties. Furthermore, its pH-resiliency relates to the neutral state favored by H17. Overall, these results provide mechanistic insights into how differences in the histidine content and amphipathicity of peptides can elicit different directionality of membrane insertion and pH-dependent permeabilization. This work features complementary methods, including dye leakage assays, NMR-monitored titrations, X-ray and neutron diffraction, oriented CD, molecular dynamics, electrochemical impedance spectroscopy, surface plasmon resonance, and quartz crystal microbalance with dissipation.

In this work, quartz crystal microbalance with dissipation (QCM-D) was employed to study the kinetic processes involved in the interaction of proteins with self-assembled monolayers (SAMs) of multimodal (MM) ligands. SAMs were fabricated to mimic two chromatographic multimodal resins with varying accessibility of the aromatic moiety to provide a well-defined model system. Kinetic parameters were determined for two different proteins in the presence of the arginine and guanidine and a comparison was made with chromatographic retention data. The results indicated that the accessibility of the ligand's aromatic moiety can have an important impact on the kinetics and chromatographic retention behavior. Interestingly, arginine and guanidine had very different effects on the protein adsorption and desorption kinetics in these MM systems. For cytochrome C, arginine resulted in a significant decrease and increase in the adsorption and desorption rates, respectively, while guanidine produced a dramatic increase in the desorption rate, with minimal effect on the adsorption rate. In addition, at different concentrations of arginine, two distinct kinetic scenarios were observed. For α-chymotrypsin, the presence of 0.1 M guanidine in the aromatic exposed ligand system produced an increase in the adsorption rate and only a moderate increase in the desorption rate, which helped to explain the surprising increase in the chromatographic salt elution concentration. These results demonstrate that protein adsorption kinetics in the presence of different mobile phase modifiers and MM ligand chemistries can play an important role in contributing to selectivity in MM chromatography.

The discoveries that non-native proteins have a role in amyloidosis and that multiple protein misfolding diseases can occur concurrently suggest that cross-seeding of amyloidogenic proteins may be central to misfolding. To study this process, a synthetic chimeric amyloidogenic protein (YEHK21-YE8) composed of two components, one that readily folds to form fibrils (YEHK21) and one that does not (YE8), was designed. Secondary structural conformational changes during YEHK21-YE8 aggregation demonstrate that, under the appropriate conditions, YEHK21 is able to induce fibril formation of YE8. The unambiguous demonstration of the induction of folding and fibrillation within a single molecule illuminates the factors controlling this process and hence suggests the importance of those factors in amyloidogenic diseases.

In order to measure the intermolecular binding forces between two halves (or partners) of naturally split protein splicing elements called inteins, a novel thiol-hydrazide linker was designed and used to orient immobilized antibodies specific for each partner. Activation of the surfaces was achieved in one step, allowing direct intermolecular force measurement of the binding of the two partners of the split intein (called protein trans-splicing). Through this binding process, a whole functional intein is formed resulting in subsequent splicing. Atomic force microscopy (AFM) was used to directly measure the split intein partner binding at 1 μm/s between native (wild-type) and mixed pairs of C- and N-terminal partners of naturally occurring split inteins from three cyanobacteria. Native and mixed pairs exhibit similar binding forces within the error of the measurement technique (~52 pN). Bioinformatic sequence analysis and computational structural analysis discovered a zipper-like contact between the two partners with electrostatic and nonpolar attraction between multiple aligned ion pairs and hydrophobic residues. Also, we tested the Jarzynski's equality and demonstrated, as expected, that nonequilibrium dissipative measurements obtained here gave larger energies of interaction as compared with those for equilibrium. Hence, AFM coupled with our immobilization strategy and computational studies provides a useful analytical tool for the direct measurement of intermolecular association of split inteins and could be extended to any interacting protein pair.

The antiallergy and potential anticancer drug tranilast has been patented for treating Alzheimer's disease (AD), in which amyloid β-protein (Aβ) plays a key pathogenic role. We used solution NMR to determine that tranilast binds to Aβ40 monomers with ∼300 μM affinity. Remarkably, tranilast increases Aβ40 fibrillation more than 20-fold in the thioflavin T assay at a 1:1 molar ratio, as well as significantly reducing the lag time. Tranilast likely promotes fibrillation by shifting Aβ monomer conformations to those capable of seed formation and fibril elongation. Molecular docking results qualitatively agree with NMR chemical shift perturbation, which together indicate that hydrophobic interactions are the major driving force of the Aβ-tranilast interaction. These data suggest that AD may be a potential complication for tranilast usage in elderly patients.

Abeta binding alcohol dehydrogenase (ABAD) is an NAD-dependent mitochondrial dehydrogenase. The binding between ABAD and Abeta is likely a direct link between Abeta and mitochondrial toxicity in Alzheimer's disease. In this study, surface plasmon resonance (SPR) was employed to determine the temperature dependence of the affinity of the ABAD-Abeta interaction. A van't Hoff analysis revealed that the ABAD-Abeta association is driven by a favorable entropic change (DeltaS = 300 +/- 30 J mol-1 K-1) which overcomes an unfavorable enthalpy change (DeltaH = 49 +/- 7 kJ/mol). Therefore, hydrophobic interactions and changes in protein dynamics are the dominant driving forces of the ABAD-Abeta interaction. This is the first dissection of the entropic and enthalpic contribution to the energetics of a protein-protein interaction involving Abeta. SPR confirmed the conformational changes in the ABAD-Abeta complex after Abeta binding, consistent with differences seen in the crystal structures of free ABAD and the ABAD-Abeta complex. Saturation transfer difference (STD) NMR experiments directly and unambiguously demonstrated the inhibitory effect of Abeta on the ABAD-NAD interaction. Conversely, NAD inhibits the Abeta-ABAD interaction. Binding of Abeta and binding of NAD to ABAD are likely mutually exclusive. Thus, Abeta binding induces conformational and subsequently functional changes in ABAD, which may have a role in the mechanism of Abeta toxicity in Alzheimer's disease.