Targeting Iron - Respiratory Reciprocity Promotes Bacterial Death.

Discovering new bacterial signaling pathways offers unique antibiotic strategies. Here, through an unbiased resistance screen of 3,884 gene knockout strains, we uncovered a previously unknown non-lytic bactericidal mechanism that sequentially couples three transporters and downstream transcription to lethally suppress respiration of the highly virulent P. aeruginosa strain PA14 - one of three species on the WHO's 'Priority 1: Critical' list. By targeting outer membrane YaiW, cationic lacritin peptide 'N-104' translocates into the periplasm where it ligates outer loops 4 and 2 of the inner membrane transporters FeoB and PotH, respectively, to suppress both ferrous iron and polyamine uptake. This broadly shuts down transcription of many biofilm-associated genes, including ferrous iron-dependent TauD and ExbB1. The mechanism is innate to the surface of the eye and is enhanced by synergistic coupling with thrombin peptide GKY20. This is the first example of an inhibitor of multiple bacterial transporters.

Tuning Structural Defects on a Nominal Single-Layered Graphene Oxide Membrane for Selective Separation of Biomolecules.

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.

Virucidal N95 Respirator Face Masks via Ultrathin Surface-Grafted Quaternary Ammonium Polymer Coatings.

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, C12-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.

Highly Sensitive Immuno-CRISPR Assay for CXCL9 Detection.

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.

Cell-free production of isobutanol: A completely immobilized system.

A completely immobilized cell-free enzyme reaction system was used to convert ketoisovaleric acid to isobutanol, a desirable biofuel, with a molar yield of 43% and a titer of 2 g/L, which are comparable to high performing in vivo systems (e.g. 41% and 5.4 g/L, respectively, for Clostridium thermocellum). The approach utilizes, for the first time, a series of previously reported enzyme mutants that either overproduce the product or are more stable when compared with their wild type. The selected enzyme variants include keto-acid decarboxylase attached to a maltose binding protein, alcohol dehydrogenase, and formate dehydrogenase. These enzymes were screened for thermal, pH, and product stability to choose optima for this system which were pH 7.4 and 35 °C. This system is designed to address well-known limitations of in vivo systems such as low product concentrations due to product feedback inhibition, instability of cells, and lack of economic product recovery.

Interactions of nuclear transport factors and surface-conjugated FG nucleoporins: Insights and limitations.

Protein-protein interactions are central to biological processes. In vitro methods to examine protein-protein interactions are generally categorized into two classes: in-solution and surface-based methods. Here, using the multivalent interactions between nucleocytoplasmic transport factors and intrinsically disordered FG repeat containing nuclear pore complex proteins as a model system, we examined the utility of three surface-based methods: atomic force microscopy, quartz crystal microbalance with dissipation, and surface plasmon resonance. Although results were comparable to those of previous reports, the apparent effect of mass transport limitations was demonstrated. Additional experiments with a loss-of-interaction FG repeat mutant variant demonstrated that the binding events that take place on surfaces can be unexpectedly complex, suggesting particular care must be exercised in interpretation of such data.

Structure and Function in Antimicrobial Piscidins: Histidine Position, Directionality of Membrane Insertion, and pH-Dependent Permeabilization.

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.

Detection of amyloid ß oligomers toward early diagnosis of Alzheimer's disease.

Amyloid ß (Aß) peptide accumulation in the brain is considered to be one of the hallmarks of Alzheimer's disease. Here, we compare two analytical techniques for detecting neurotoxic Aß1-42 oligomers - Quartz Crystal Microbalance with Dissipation (QCM-D) and Single Molecule Array (Simoa). Both detection methods exploit a feature of the monoclonal antibody bapineuzumab, which targets N-terminal residues 1-5 of Aß with high affinity and use it as both a capture and detection reagent. Assays developed with the two methods allow us to specifically recognize neurotoxic Aß1-42 oligomers and higher aggregates such as fibrils but discriminate against Aß1-42 monomer species. We find that for detection of Aß1-42 oligomers, Simoa was roughly 500 times more sensitive than the QCM-D technique with limits of detection of 0.22 nM and 125 nM, respectively.

Oriented chiral water wires in artificial transmembrane channels.

Aquaporins (AQPs) feature highly selective water transport through cell membranes, where the dipolar orientation of structured water wires spanning the AQP pore is of considerable importance for the selective translocation of water over ions. We recently discovered that water permeability through artificial water channels formed by stacked imidazole I-quartet superstructures increases when the channel water molecules are highly organized. Correlating water structure with molecular transport is essential for understanding the underlying mechanisms of (fast) water translocation and channel selectivity. Chirality adds another factor enabling unique dipolar oriented water structures. We show that water molecules exhibit a dipolar oriented wire structure within chiral I-quartet water channels both in the solid state and embedded in supported lipid bilayer membranes (SLBs). X-ray single-crystal structures show that crystallographic water wires exhibit dipolar orientation, which is unique for chiral I-quartets. The integration of I-quartets into SLBs was monitored with a quartz crystal microbalance with dissipation, quantizing the amount of channel water molecules. Nonlinear sum-frequency generation vibrational spectroscopy demonstrates the first experimental observation of dipolar oriented water structures within artificial water channels inserted in bilayer membranes. Confirmation of the ordered confined water is obtained via molecular simulations, which provide quantitative measures of hydrogen bond strength, connectivity, and the stability of their dipolar alignment in a membrane environment. Together, uncovering the interplay between the dipolar aligned water structure and water transport through the self-assembled I-quartets is critical to understanding the behavior of natural membrane channels and will accelerate the systematic discovery for developing artificial water channels for water desalting.

Protein Binding Kinetics in Multimodal Systems: Implications for Protein Separations.

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.

A2T and A2V Aß peptides exhibit different aggregation kinetics, primary nucleation, morphology, structure, and LTP inhibition.

The histopathological hallmark of Alzheimer's disease (AD) is the aggregation and accumulation of the amyloid beta peptide (Aß) into misfolded oligomers and fibrils. Here we examine the biophysical properties of a protective Aß variant against AD, A2T, and a causative mutation, A2T, along with the wild type (WT) peptide. The main finding here is that the A2V native monomer is more stable than both A2T and WT, and this manifests itself in different biophysical behaviors: the kinetics of aggregation, the initial monomer conversion to an aggregation prone state (primary nucleation), the abundances of oligomers, and extended conformations. Aggregation reaction modeling of the conversion kinetics from native monomers to fibrils predicts the enhanced stability of the A2V monomer, while ion mobility spectrometry-mass spectrometry measures this directly confirming earlier predictions. Additionally, unique morphologies of the A2T aggregates are observed using atomic force microscopy, providing a basis for the reduction in long term potentiation inhibition of hippocampal cells for A2T compared with A2V and the wild type (WT) peptide. The stability difference of the A2V monomer and the difference in aggregate morphology for A2T (both compared with WT) are offered as alternate explanations for their pathological effects.

Accelerated insulin aggregation under alternating current electric fields: Relevance to amyloid kinetics.

The time-dependent nucleation phase is critical to amyloid fibrillation and related to many pathologies, in which the conversion from natively folded amyloidogenic proteins to oligomers via nucleation is often hypothesized as a possible underlying mechanism. In this work, non-uniform AC-electric fields across two asymmetric electrodes were explored to control and examine the aggregation of insulin, a model amyloid protein, in aqueous buffer solution at constant temperature (20 °C) by fluorescence correlation spectroscopy and fluorescence microscopy. Insulin was rapidly concentrated in a strong AC-field by imposed AC-electroosmosis flow over an optimal frequency range of 0.5-2 kHz. In the presence of an AC-field, direct fibrillation from insulin monomers without the formation of oligomer precursors was observed. Once the insulin concentration had nearly doubled its initial concentration, insulin aggregates were observed in solution. The measured lag time for the onset of insulin aggregation, determined from the abrupt reduction in insulin concentration in solution, was significantly shortened from months or years in the absence of AC-fields to 1 min-3 h under AC-fields. The ability of external fields to alter amyloid nucleation kinetics provides insights into the onset of amyloid fibrillation.

Chimera-induced folding: implications for amyloidosis.

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.

Egg white varnishes on ancient paintings: a molecular connection to amyloid proteins.

For about 400 years, egg white was used to coat and protect paintings without detailed understanding of its molecular properties. A molecular basis is provided for its advantageous properties and one of its protective properties is demonstrated with oxygen transport behavior. Compared to the native secondary structure of ovalbumin in solution of circa 33% α-helix and ß-sheet, attenuated total reflection-FTIR (ATR-FTIR) spectra showed a 73% decrease of α-helix content and a 44% increase of ß-sheet content over eight days. The data suggest that the final coating of dissolved ovalbumin from egg white after long exposure to air, which is hydrophobic, comprises mostly ß-sheet content (ca. 50%), which is predicted to be the lowest-energy structure of proteins and close to that found in amyloid fibrils. Coating a synthetic polytetrafluoroethylene membrane with multiple layers of egg white decreased oxygen diffusion by 50% per layer with a total decrease of almost 100% for four layers.

Detection and structural characterization of insulin prefibrilar oligomers using surface enhanced Raman spectroscopy.

In vitro fibril formation typically exhibits a lag phase followed by a rapid elongation phase. Soluble prefibrilar oligomers form as multiple assembly states occur during the lag phase and, after forming a nucleus, rapidly propagate into amyloid aggregates and fibrils. The structure and morphology of amyloid fibrils have been extensively characterized over the last decades, while little is known about the structural organization of the prefibrilar oligomers or their multiple assembly states. The main difficulty in structural characterization of prefibrilar aggregates is their low concentration (pmolar) and their continual reactive conversion. Herein we overcome these difficulties by utilizing Surface-Enhanced Raman Spectroscopy (SERS) with a model amyloid peptide, insulin. SERS is a powerful analytic tool that is able to provide detection of small molecules down to a single-molecule level. Using SERS we found that during the 3 lag phase before the onset of insulin fibril formation, the amount of insulin oligomers increased more than twice after the first hour of incubation under fibrillation conditions (pH 1.6, 65°C) and then slowly decreased with time. The latter finding is kinetically linked to the conversion of the prefibrilar oligomers into fibril species. This study provides valuable new information about the time-dependent structural organization of insulin oligomers and demonstrates the power and potential of SERS for detection and structural characterization of biological specimens present at low concentrations.

Oriented covalent immobilization of antibodies for measurement of intermolecular binding forces between zipper-like contact surfaces of split inteins.

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.

Tranilast binds to aß monomers and promotes aß fibrillation.

The antiallergy and potential anticancer drug tranilast has been patented for treating Alzheimer's disease (AD), in which amyloid ß-protein (Aß) plays a key pathogenic role. We used solution NMR to determine that tranilast binds to Aß40 monomers with ∼300 µM affinity. Remarkably, tranilast increases Aß40 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 Aß 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 Aß-tranilast interaction. These data suggest that AD may be a potential complication for tranilast usage in elderly patients.

A multi-dimensional approach for fractionating proteins using charged membranes.

In an effort to increase selectivity among proteins with crossflow ultrafiltration, we offer and demonstrate a comprehensive approach to fractionate proteins of similar molecular weight and relatively close pI values. This multidimensional approach involves optimizing membrane charge type and density together with operating conditions such as precise control of pH, ionic strength, and transmembrane pressure for reduced membrane fouling. Each filtration experiment was performed in cross-flow configuration for ∼20 min, allowing fast screening for optimal separation as determined by maximum selectivity, Ψ, and purity, P. Using our comprehensive approach for fractionating mixtures RNase A-lysozyme and BSA-hemoglobin, we obtained values of Ψ = 9.1, P = 95.7%, and Ψ = 6.5, P = 62.1%, respectively.

Evaluating nuclei concentration in amyloid fibrillation reactions using back-calculation approach.

BACKGROUND: In spite of our extensive knowledge of the more than 20 proteins associated with different amyloid diseases, we do not know how amyloid toxicity occurs or how to block its action. Recent contradictory reports suggest that the fibrils and/or the oligomer precursors cause toxicity. An estimate of their temporal concentration may broaden understanding of the amyloid aggregation process. METHODOLOGY/PRINCIPAL FINDINGS: Assuming that conversion of folded protein to fibril is initiated by a nucleation event, we back-calculate the distribution of nuclei concentration. The temporal in vitro concentration of nuclei for the model hormone, recombinant human insulin, is estimated to be in the picomolar range. This is a conservative estimate since the back-calculation method is likely to overestimate the nuclei concentration because it does not take into consideration fibril fragmentation, which would lower the amount of nuclei CONCLUSIONS: Because of their propensity to form aggregates (non-ordered) and fibrils (ordered), this very low concentration could explain the difficulty in isolating and blocking oligomers or nuclei toxicity and the long onset time for amyloid diseases.

Isolating toxic insulin amyloid reactive species that lack ß-sheets and have wide pH stability.

Amyloid diseases, including Alzheimer's disease, are characterized by aggregation of normally functioning proteins or peptides into ordered, ß-sheet rich fibrils. Most of the theories on amyloid toxicity focus on the nuclei or oligomers in the fibril formation process. The nuclei and oligomers are transient species, making their full characterization difficult. We have isolated toxic protein species that act like an oligomer and may provide the first evidence of a stable reactive species created by disaggregation of amyloid fibrils. This reactive species was isolated by dissolving amyloid fibrils at high pH and it has a mass >100 kDa and a diameter of 48 ± 15 nm. It seeds the formation of fibrils in a dose dependent manner, but using circular dichroism and deep ultraviolet resonance Raman spectroscopy, the reactive species was found to not have a ß-sheet rich structure. We hypothesize that the reactive species does not decompose at high pH and maintains its structure in solution. The remaining disaggregated insulin, excluding the toxic reactive species that elongated the fibrils, returned to native structured insulin. This is the first time, to our knowledge, that a stable reactive species of an amyloid reaction has been separated and characterized by disaggregation of amyloid fibrils.