Template Protein Research Articles

From stars to stripes: RNA-directed shaping of plant viral protein templates-structural synthetic virology for smart biohybrid nanostructures.

The self-assembly of viral building blocks bears exciting prospects for fabricating new types of bionanoparticles with multivalent protein shells. These enable a spatially controlled immobilization of functionalities at highest surface densities-an increasing demand worldwide for applications from vaccination to tissue engineering, biocatalysis, and sensing. Certain plant viruses hold particular promise because they are sustainably available, biodegradable, nonpathogenic for mammals, and amenable to in vitro self-organization of virus-like particles. This offers great opportunities for their redesign into novel "green" carrier systems by spatial and structural synthetic biology approaches, as worked out here for the robust nanotubular tobacco mosaic virus (TMV) as prime example. Natural TMV of 300 x 18 nm is built from more than 2,100 identical coat proteins (CPs) helically arranged around a 6,395 nucleotides ssRNA. In vitro, TMV-like particles (TLPs) may self-assemble also from modified CPs and RNAs if the latter contain an Origin of Assembly structure, which initiates a bidirectional encapsidation. By way of tailored RNA, the process can be reprogrammed to yield uncommon shapes such as branched nanoobjects. The nonsymmetric mechanism also proceeds on 3'-terminally immobilized RNA and can integrate distinct CP types in blends or serially. Other emerging plant virus-deduced systems include the usually isometric cowpea chlorotic mottle virus (CCMV) with further strikingly altered structures up to "cherrybombs" with protruding nucleic acids. Cartoon strips and pictorial descriptions of major RNA-based strategies induct the reader into a rare field of nanoconstruction that can give rise to utile soft-matter architectures for complex tasks. This article is categorized under: Biology-Inspired Nanomaterials > Protein and Virus-Based Structures Nanotechnology Approaches to Biology > Nanoscale Systems in Biology Biology-Inspired Nanomaterials > Nucleic Acid-Based Structures.

Improvement of Adsorption Selectivity of Ion-Exchange Chromatography through the Double-Recognition Mode

Ion-exchange chromatography (IEC) was combined with molecular imprinting technology (MIT) to prepare two kinds of double-recognition resins (DEAE@BSA and DEAE@bIgG) using bovine serum albumin (BSA) and bovine immunoglobulin G (bIgG) as the imprinting template proteins. New resins consist of ion-exchange interaction and spatial recognition and were characterized by Fourier transform infrared, scanning electron microscopy–energy-dispersive X-ray spectrometry, thermogravimetric analysis, and Brunauer–Emmett–Teller techniques. The static and dynamic adsorption behaviors of proteins on resins were investigated. In addition, the competitive adsorption between BSA and bIgG was also studied. It was found that the adsorbed protein density (Qc) of the target protein remained the same after imprinting, but the competitive protein dropped obviously. The selectivity factors (α) of DEAE@BSA and DEAE@bIgG could reach 3.56 and 2.56, respectively. The results of adsorption kinetics and frontal adsorption experiments furth...

Production of Multicomponent Protein Templates for the Positioning and Stabilization of Enzymes.

Harnessing the ability of proteins to self-assemble into complex structures has enabled the creation of templates for applications in nanotechnology. Protein templates can be used to position functional molecules in regular patterns with nanometer precision over large surface areas. A difficult but successful approach to building customizable protein templates involves designing novel protein-protein interfaces to join protein building blocks into ordered arrangements. This approach was illustrated recently by engineering the protein interfaces of a molecular chaperone to produce filamentous templates composed of repeating subunits. In this chapter, we describe how these multicomponent protein templates can be produced recombinantly, assembled into filaments, and used as material templates. The templates enable the positioning and alignment of functional molecules at varying distances along the length of the filament, which can be demonstrated using a Förster resonance energy transfer (FRET) assay. In addition, we describe a method to quantify the chaperone ability of these filaments to stabilize and protect other proteins from thermal-induced aggregation-a useful property for bionanotechnology applications that involve molecular scaffolds for positioning and stabilizing enzymes.

Preparation of bovine hemoglobin surface molecularly imprinted cotton for selective protein recognition

Abstract We have developed a bovine hemoglobin (BHb) surface molecularly imprinted cotton based on degreasing cotton via surface imprinting technique for the efficient selective adsorption of BHb. The morphological structure of the samples was characterized by Scanning Electron Microscopy (SEM), and the chemical modification steps were characterized by Fourier Transform Infrared Spectroscopy (FTIR). The maximum adsorption capacity of the molecularly imprinted cotton (MIC) and non-imprinted cotton (NIC) for BHb was 62.95 mg/g and 8.32 mg/g, respectively, at the optimum pH value of 6.2. The kinetics studies demonstrated that the adsorption follows a pseudo-first-order kinetic model. The adsorption isotherm analysis indicated that the Langmuir adsorption isotherm fits well with the adsorption equilibrium data. Also, the selective adsorption shows the MIC has a good selectivity for BHb. In addition, the assessment of the reusability of the MIC was tested for five successive cycles revealed no significant decrease of the adsorption capacity. Electrophoretic analysis suggests the MIC were successfully applied to capture template proteins from the bovine blood sample.

Decoding the signature of molecular mechanism involved in mutation associated resistance to 1, 3-benzothiazin-4-ones (Btzs) based DprE1 inhibitors using BTZ043 as a reference drug

ABSTRACTDifferent resistant strains of M. tuberculosis (Mtb) highlight the urgent need of novel anti-tubercular drugs. In mycobacteria, decaprenyl-phosphoryl-β-D-ribose 2’-oxidase (DprE1) is an appealing enzyme to target as it is involved in the biosynthesis of cell wall component arabinogalactan.1, 3-benzothiazin-4-ones (BTZs) based drugs are promising irreversible inhibitors of DprE1. However, a single point mutation of Cys387Ser in DprE1 results in the development of resistance to these drugs. Herein, we made an effort to decode the molecular mechanism of Cys387Ser DprE1 mutation associated resistance in Mtb against BTZs using different in silico techniques. Since the 3D crystal structure of mutant Cys387Ser protein is not yet been solved, thus the homology model was also developed using 4P8N as a template protein with 99.8% homology with the target protein. The computational results suggested that the factors like HOMO–LUMO energy gap, Burgi-Dunitz angle and distance support the covalent inhibition of wild DprE1 by 1, 3-benzothiazin-4-ones class of drugs, using BTZ043 as a reference drug and the same factors support the cause of resistance in case of Cys387Ser mutation. On the basis of these results, it was concluded that BTZ043 can efficiently inhibit the wild type DprE1 than mutant DprE1.

Preparation of molecularly imprinted polymers on hemin-graphene surface for recognition of high molecular weight protein.

Surface molecular imprinting for proteins, especially for the high molecular weight protein, are facing a great challenge today. We report a novel design to prepare molecular imprinting polymers (MIPs) on the surface of hemin-graphene nanosheets (H-GNs) for the recognition of thyroglobulin (Tg, 660 KD). H-GNs with an intrinsic peroxidase-like activity could effectively immobilize the protein template on its surface to improve the number of imprinted sites per unit surface area and then directly initiate free-radical polymerization to synthesize MIPs. The results indicated MIPs film could be prepared on the surface of H-GNs/filter paper, implying the advantages of surface imprinting, and exhibiting high adsorption capacity (400 mg g-1). The MIP composites clearly distinguished the proteins on the basis of the synergistic effect of shape complementarity and multiple interactions, not even anti-Tg antibodies and sample matrix. The gray intensity of the colorimetric paper-based sensor proved to be proportional to the concentration of Tg in the range of 5-100 ng mL-1 with a detection limit of 1 ng mL-1 (15 fM). The design and direct synthesis of MIPs on the H-GNs/filter paper provide a new perspective for the surface imprinting materials with potential in the recognition and detection of proteins.

A graphene based–biomimetic molecularly imprinted polyaniline sensor for ultrasensitive detection of human cardiac troponin T (cTnT)

Abstract In the present work, a biomimetic nano-molecularly imprinted polymer (N-MIP) electrode based on a graphene screen-printed electrode was developed for the ultrasensitive detection of cardiac troponin T (cTnT). The biomimetic cavities for targeted sensing for analyte were fabricated by the electropolymerization of conductive co-polymer matrix of aniline and carboxylated aniline on the graphene oxide (GO) electrode, in the presence template protein (cTnT for cardiac troponin T probe) by cyclic voltammetry. The surface characterization of the sensor was performed using cyclic voltammetry (CV) and differential pulse voltammetry (DPV), and scanning electron microscopy (SEM). The best biomimetic surface nanotexture was obtained at aniline/carboxylatedaniline ratio of 1:4. The linear range of cTnT probe was in the range of 0.02 to 0.09 ng/mL, with the detection limit of 0.008 ng/mL. The reliability of the N-MIP cTnT sensor was examined by comparing the results with those obtained from HPLC method, and it was observed that the results from N-MIP sensors and HPLC have a great correlation.

Ionic liquids skeleton typed magnetic core-shell molecularly imprinted polymers for the specific recognition of lysozyme

The novel ionic liquids skeleton typed magnetic core-shell molecularly imprinted polymers (Fe3O4–COOH@IL-MIP) were firstly constructed with 1-vinyl-3-aminoformylmethyl imidazolium chloride ionic liquid ([VAFMIM]Cl-IL) modified magnetic particles as the substrate materials, [VAFMIM]Cl-IL as functional monomer, 1,6-hexanediyl-3,3′-bis-1-vinylimidazolium dichloride ionic liquid as cross-linker and Lysozyme (Lys) as template protein via surface-imprinting technique. The structure of Fe3O4–COOH@IL-MIP were confirmed by transmission and scanning electron microscopy, dynamic light scattering, thermo-gravimetric analysis, fourier transform infrared spectrometry and X-ray diffraction. The adsorption mechanism was discussed from the perspective of amino acid residues of Lys. The maximum adsorption capacity of MIPs was 166.36 mg g−1 and imprinting factor was 2.67. The competitive adsorption experiments demonstrated the favorable recognition ability of MIPs toward Lys. Reusability studies indicated MIPs can be reused ten times without obvious loss of rebinding ability. The Lys conformation maintained intact after elution and the elution rate was as high as 74%. The adsorption experiment of egg white manifested that MIPs can effectively separate Lys in practical samples. Only ILs and Fe3O4 were utilized to fabricate MIPs, this strategy realized the goal of energy and cost saving while achieving simple synthesis of imprinted materials, and is expected to provide a new feasible idea to exploit synthetic methods for protein-MIPs.

Design and site-directed compartmentalization of gold nanoclusters within the intrasubunit interfaces of ferritin nanocage

BackgroundProtein nanocages have emerged as popular nanocarriers for either drug delivery or biotemplates for the preparation of nanomaterials. However, only three interfaces, namely exterior surface, intersubunit and inner cavity, have been used as reaction sites for the above purposes with all known protein nanocages. On the other hand, how to control the site of Au NCs formed within a targeted protein template while maintaining the functionality of protein itself remains challenging.ResultsIn this work, inspired by compartmentalization in living systems, we firstly come up with the conception of “intrasubunit interfaces”, located within subunit of protein nanocage. We built a new, specific compartment for fabrication of gold nanoclusters by genetic modification of the inherent ferroxidase center located within four-α-helix bundle of each ferritin subunit. This newly built compartment not only realizes the site-directed synthesis of gold nanoclusters but also has no effect on the functionality of ferritin itself such as encapsulation by its inner cavity. These redesigned composites can be further applied as fluorescent imaging agent and carriers for preparation of hybrid nanomaterials.ConclusionsThe designing strategy of intrasubunit interfaces opens a new way for future applications of cage-like proteins.

Adsorption-selectivity customization and competitive adsorption of tryptamine-based mixed-mode chromatography

Mixed-mode chromatography (MMC) shows great promise for applications in protein purification. Therefore, novel MMC ligands are always desired. However, achieving high adsorption selectivity of ligands is a major challenge due to competitive adsorption. Herein, MMC with tryptamine (4 F F-TA) exhibited equal adsorption capacity for bovine serum albumin (BSA) and bovine immunoglobulin (bIgG), affording a unique competitive adsorption process, but non ideal purity. Molecular imprinting technology (MIT) was adopted for the customized adsorption selectivity of MMC, and BSA and bIgG were used as imprinting templates. Static and dynamic binding performances under competitive adsorption before and after imprinting were investigated at different pH values, salt concentrations, and BSA/bIgG mass ratios. The results showed that the adsorption capacity of the target protein remained similar level but competitive protein adsorption dropped significantly (below 40 mg/g resin). This indicated that the adsorption selectivity of 4 F F-TA could be customized by changing the template protein used during imprinting. In addition, the pH-dependence and salt tolerance of MMC were unchanged after imprinting. The binding behaviors of BSA and bIgG in column chromatographic separation further confirmed the improved in adsorption selectivity. The purity of BSA increased from 55.4% to 78.2% for 4 F F-TA@BSA and that of bIgG from 40.7% to 71.2% for 4 F F-TA@bIgG. This study indicated that MIT is an effective strategy to change and improve the adsorption selectivity of MMC.

Analysis of the Full-Length Pyriform Spidroin Gene Sequence.

Spiders often produce multiple types of silk, each with unique properties suiting them to certain tasks and biological functions. Orb-weaver spiders can generate more than six types of silk fibroins, with pyriform silk used to form attachment discs, adhering silk to other surfaces and substances. The unique higher-order structuring of silk fibroins has been cited as the source of their remarkable biomechanical properties. Even so, only one full-length gene sequence of pyriform silk protein 1 (PySp1) from Argiopeargentata has been reported, and studies on the mechanical properties of natural pyriform silk fibers are also lacking. To better understand the PySp1 family of genes, we used long-distance PCR (LD-PCR) to determine the sequence of PySp1 in the Araneusventricosus species. This full-length PySp1 gene is 11,931 bp in length, encoding for 3976 amino acids residues in non-repetitive N- and C-terminal domains with a central largely repetitive region made up of sixteen remarkably homogeneous units. This was similar to the previously reported A. argentata PySp1 sequence, with PySp1 from A. ventricosus also having a long repetitive N-linker that bridges the N-terminal and repetitive regions. Predictions of secondary structure and hydrophobicity of A. ventricosus PySp1 showed the pyriform silk fiber’s functional properties. The amino acid compositions of PySp1 is obviously distinct from other spidroins. Our sequence makes an important contribution to understand pyriform silk protein structure and also provides a new template for recombinant pyriform silk proteins with attractive properties.

Multifunctional oligomer immobilized on quartz crystal microbalance: a facile and stabilized molecular imprinting strategy for glycoprotein detection

Glycoprotein detection holds great potential for early diagnosis of diverse diseases. For this purpose, the combination of quartz crystal microbalance (QCM) sensor and molecular imprinting has attracted increasing attention. Nonetheless, the recently common imprinted films fabricated on QCM electrode are thick and rigid, lacking flexibility in aqueous phase. Alternatively, small molecules immobilized on the electrode to construct molecular scale film could address this problem, while stabilization of the imprinted sites remains challenging. Herein, a co-assembly complex was obtained by the mixture of template and multifunctional oligomer, which was then immobilized on the amino-modified transducer surface through epoxy-amino reaction to form a protein-imprinted film. Afterward, the remaining epoxy groups in oligomer chains were cross-linked to conserve and stabilize the orientation of imprinted sites after template elution. Template rebinding tests show that cross-linked film has much higher imprinting factors than that of the non-cross-linked counterpart. Furthermore, control proteins that are distinct in properties and structures were employed to demonstrate the selectivity of this approach, and the imprinted assay reveals high affinity and specificity towards template protein. Graphical Abstract.

High-throughput targeted gene deletion in the model mushroom Schizophyllum commune using pre-assembled Cas9 ribonucleoproteins

Efficient gene deletion methods are essential for the high-throughput study of gene function. Compared to most ascomycete model systems, gene deletion is more laborious in mushroom-forming basidiomycetes due to the relatively low incidence of homologous recombination (HR) and relatively high incidence of non-homologous end-joining (NHEJ). Here, we describe the use of pre-assembled Cas9-sgRNA ribonucleoproteins (RNPs) to efficiently delete the homeodomain transcription factor gene hom2 in the mushroom-forming basidiomycete Schizophyllum commune by replacing it with a selectable marker. All components (Cas9 protein, sgRNA, and repair template with selectable marker) were supplied to wild type protoplasts by PEG-mediated transformation, abolishing the need to optimize the expression of cas9 and sgRNAs. A Δku80 background further increased the efficiency of gene deletion. A repair template with homology arms of 250 bp was sufficient to efficiently induce homologous recombination. This is the first report of the use of pre-assembled Cas9 RNPs in a mushroom-forming basidiomycete and this approach may also improve the genetic accessibility of non-model species.

Pregenomic RNA: How to assist the management of chronic hepatitis B?

Pregenomic RNA (pgRNA) is an emerging serological marker for chronic hepatitis B virus (HBV) infection. While pgRNA is principally the template for viral proteins and viral DNAs, additional novel functions for the serum pgRNA have recently been described. These results extend for pgRNA a regulatory function in the viral life cycle and potentially a role in pathogenesis. Here, we review the diverse roles of pgRNA in HBV replication and pathogenesis, emphasizing how the unique structure of this RNA is key to its various functions. We focus in particular on the role of HBV pgRNA in guiding antiviral therapy including nucleot(s)ide analog interruption and role as a marker of cure with new curative therapies. We also briefly allude to the emerging niche for new direct-acting or indirect-acting antivirals targeting pgRNA.

Molecularly imprinted polymer-based reusable biosensing device on stainless steel for spatially localized detection of cytokine IL-1β

A molecularly imprinted polymer (MIP) based biosensing device on stainless steel (SS) for detection of locally variable concentration of cytokine interleukin-1β (IL-1β) was successfully developed using a sandwich assay scheme. The SS surface was firstly modified with a layer of polydopamine (PDA) followed by the attachment of a layer of poly(ethyleneimine) (PEI) by electrostatic adsorption. Subsequently, the template protein IL-1β was adsorbed on the PEI terminated SS surface due to electrostatic adsorption. A PDA imprinting film was then in-situ synthesized on the surface of the modified SS substrate with incorporated template cytokine. Finally, the template was washed off the SS substrate leaving behind cavities with specific shape and capable of capturing cytokines thus forming a MIP biosensing interface. After exposure to the analyte IL-1β, the MIP biosensing device was incubated with IL-1β detection antibody-modified fluorescent polystyrene beads allowing to determine the amount of captured IL-1β based on fluorescence intensity. The device has been demonstrated to detect IL-1β with low detection limit of 10.2 pg mL−1, and a linear detection range of 25–400 pg mL−1. This MIP biosensing device can be regenerated more than three times with coefficient of variation 2.08%. The device was applied for the detection of IL-1β secreted by rat macrophages, where the good specificity and selectivity were achieved. MIP serves in this device as a superior substitute of antibody with exceptional stability and reusability. The MIP based biosensing technology presented in our work paves a new way for developing a universal and robust sensing platform for the detection of spatially localised small proteins with low physical concentration.

A novel series of M(II) complexes of 6‐methylpyridine‐2‐carboxylic acid with 4(5)methylimidazole: Synthesis, crystal structures, α‐glucosidase activity, density functional theory calculations and molecular docking

Novel complexes of 6‐methylpyridine‐2‐carboxylic acid and 4(5)methylimidazole, namely [Mn(6‐mpa)2(4(5)MeI)2] (1), [Zn(6‐mpa)2(4(5)MeI)2] (2), [Cd(6‐mpa)2(4(5)MeI)2] (3), [Co(6‐mpa)2(4(5)MeI)2] (4), [Ni(6‐mpa)2(4(5)MeI)(OAc)] (5) and [Cu(6‐mpa)2(4(5)MeI)] (6), were synthesized for the first time. The structures of complexes 1–4 and complexes 5 and 6 were determined using X‐ray diffraction and mass spectrometric techniques, respectively. The experimental spectral analyses for these complexes were performed using Fourier transform infrared and UV–visible techniques. The α‐glucosidase inhibition activity values (IC50) of complexes 1–6 were identified in view of genistein reference compound. Moreover, the DFT/HSEh1PBE/6‐311G(d,p)/LanL2DZ level was used to obtain optimal molecular geometry and vibrational wavenumbers for complexes 1–6. Electronic spectral behaviours and major contributions to the electronic transitions were investigated using TD‐DFT/HSEh1PBE/6‐311G(d,p)/LanL2DZ level with conductor‐like polarizable continuum model and SWizard program. Finally, in order to investigate interactions between the synthesized complexes (1–6) and target protein (template structure S. cerevisiae isomaltase), a molecular docking study was carried out.

Fixed DNA Molecule Arrays for High-Throughput Single DNA-Protein Interaction Studies.

The DNA Curtains assay is a recently developed experimental platform for protein-DNA interaction studies at the single-molecule level that is based on anchoring and alignment of DNA fragments. The DNA Curtains so far have been made by using chromium barriers and fluid lipid bilayer membranes, which makes such a specialized assay technically challenging and relatively unstable. Herein, we report on an alternative strategy for DNA arraying for analysis of individual DNA-protein interactions. It relies on stable DNA tethering onto nanopatterned protein templates via high affinity molecular recognition. We describe fabrication of streptavidin templates (line features as narrow as 200 nm) onto modified glass coverslips by combining surface chemistry, atomic force microscopy (AFM), and soft lithography techniques with affinity-driven assembly. We have employed such chips for arraying single- and double-tethered DNA strands, and we characterized the obtained molecular architecture: we evaluated the structural characteristics and specific versus nonspecific binding of fluorescence-labeled DNA using AFM and total internal reflection fluorescence microscopy. We demonstrate the feasibility of our DNA molecule arrays for short single-tethered as well as for lambda single- and double-tethered DNA. The latter type of arrays proved very suitable for localization of single DNA-protein interactions employing restriction endonucleases. The presented molecular architecture and facile method of fabrication of our nanoscale platform does not require clean room equipment, and it offers advanced functional studies of DNA machineries and the development of future nanodevices.

Facile preparation of photonic and magnetic dual responsive protein imprinted nanomaterial for specific recognition of bovine hemoglobin

Stimulated response molecularly imprinted polymers (MIPs) have attracted wide attention not only because of their intriguing response to external stimuli, but also due to the desired molecular recognition ability. Herein, a novel dual responsive proteins MIPs with both photonic and magnetic stimuli has been developed by surface imprinting polymerization, where double bonds modified Fe3O4 nanoparticles, a water-soluble azobenzene containing 4-[(4-methacryloyloxy)phenylazo]benzenesulfonic acid and bovine hemoglobin (BHb) were used as magnetic core, photoswitchable functional monomer and template protein, respectively. The resultant MIPs possessed a typical spherical structure with a relative rough imprinted layer of 15 nm thickness. Moreover, the MIPs had good binding behavior toward BHb with fast binding kinetics, high adsorption capacity and satisfactory selectivity. The MIPs were proved to be well photo-controlled toward the template protein BHb under the alternating irradiation of 365 and 440 nm: 31.4% BHb was released from the MIPs at 365 nm and 67.2% BHb was bound into the MIPs at 440 nm. As a result, the MIPs were successfully applied to specifically extract template protein from actual biological sample. As a consequence, combining photocontrolled release and uptake, rapidly magnetic separation and specific recognition, the resultant MIPs with photonic and magnetic dual responsive abilities show significant advantages in the enrichment of trace protein in biological samples.

In silico Prediction of Immunogenic Sites on PMN Antigen and Design of Epitope Blocking Caps

Autoimmune disorders (AD) are a type of disease in which an individual's immune system attacks the cells of its own body. Currently, AD are the 2nd leading cause of chronic long term illness. The primary method of treating AD is the use of non‐specific immunosuppressant drugs, suppressing the entire immune system and leaving the patient vulnerable to infections. Antigen specific therapies have been proposed in order to replace treatment with immunosuppressant drugs for AD. The work here focuses on developing a new approach to AD therapy which targets the epitope site (ES) on the antigen, with an emphasis on the kidney specific AD primary membranous nephropathy, which affects 10–12 million people worldwide. Using in silico mutagenesis the ES on the phospholipase A2 receptor (PLA2R) were predicted and epitope binding proteins (EBP) were developed to target these regions. Using Epitopia and EPCES two regions on the CTLD1 domain of PLA2R were identified as potential ES. These ES are characterized in terms of the number of residues, their electrostatic potential, and their hydrophobicity. In silico prediction of immunogenicity showed the single nucleotide polymorphisms M292V, H300D, and G1106S do not directly contribute to immunogenic sites on PLA2R. In silico mutagenesis of two protein templates (5IMK and 4JE4) was performed to design EBP structures. The Rosetta protein‐protein docking server was used to evaluate the interface score of designed EBP to the PLA2R antigen, and disruption of binding to their original binding partner via molecular mechanics calculations. In silico mutations to the 5IMK monobody increased the binding interface score from −4.259 rosetta energy units (REU) to −8.903 REU. Similarly, in silico mutations to the 4JE4 monobdy increased the interface score from −3.964 REU to −5.867 REU. Data shows the introduction of charged residues to the monobody contributes to the most significant gains in interface score. Further in silico mutations were introduced to disrupt the binding preference of 5IMK and 4JE4 to their original targets. Binding potency was reduced from −7.851 REU to −3.87 REU for 5IMK, and from −12.263 REU to −3.693 REU for 4JE4. This work could be used to develop a new antigen specific therapy approach for PMN and AD in general.Support or Funding InformationWilliam Randolph Hearst FoundationThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Determining the Mechanism of Hemolytic Function and Type Vb Secretion by a Gram‐Negative Pathogen

Proteus mirabilis is a pathogenic opportunistic Gram‐negative bacterium that produces and secretes a toxic protein called hemolysin A (HpmA). P. mirabilis utilizes a type Vb, or two‐partner (TPS), secretion system to simultaneously transport and activate HpmA across its outer membrane and release it into the blood of a host where it destroys red blood cells (hemolysis). Like all other TPS proteins, HpmA contains an N‐terminal TPS domain (≈300 residues) essential for secretion and a separate C‐terminal functional domain (>1200 residues) responsible for virulent functions. Our goals are to determine how secretion of HpmA is prompted by its folding and identify regions of HpmA critical to its hemolytic function. In the absence of secretion, the unfolded full‐length HpmA requires a folded TPS domain to act as a template protein to achieve its hemolytically active form. We term these Template Assisted Hemolytic Activity (TAHA) assays. Performing TAHA assays with genetically altered HpmA, we have quantitated the importance of different regions on HpmA folding and function. We have determined the borders of the hemolytic functional domain, marking the first identification of a functional region on a TPS hemolysin. Other hemolytic proteins are known to adopt a modular method of function. To determine if the function of HpmA is dictated by similar modules, we have created small deletions and amino acid randomizations within the hemolytic functional region. Separately, by rationally designing deletions and variants within the TPS domain of full‐length HpmA, we have determined that the folded template recognizes specific sequences within the unfolded TPS domain to initiate folding. This supports our N‐terminal first secretion model and suggests a continuity of structure and linkage of folding between the TPS domain and the remainder of HpmA. A continuous β‐helical structure starting from the TPS domain could explain this link between domains of secretion and function. To test this link and characterize formation of secondary structure during templated folding, we have performed structural studies using CD spectroscopy. By identifying regions necessary for hemolysis and characterizing how TPS domain folding is required to induce hemolytic activation, our results have expanded our working model for HpmA secretion and function.Support or Funding InformationThis research was supported by an Undergraduate Research and Creativity grant and a Dean's Distinguished Summer Fellowship to GW.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.