Similarities and Specificities of Fungal Keratinolytic Proteases: Comparison of Keratinases of Paecilomyces marquandii and Doratomyces microsporus to Some Known Proteases

Based on previous screening for keratinolytic nonpathogenic fungi, Paecilomyces marquandii and Doratomyces microsporus were selected for production of potent keratinases. The enzymes were purified and their main biochemical characteristics were determined (molecular masses, optimal temperature and pH for keratinolytic activity, N-terminal amino acid sequences). Studies of substrate specificity revealed that skin constituents, such as the stratum corneum, and appendages such as nail but not hair, feather, and wool were efficiently hydrolyzed by the P. marquandii keratinase and about 40% less by the D. microsporus keratinase. Hydrolysis of keratin could be increased by the presence of reducing agents. The catalytic properties of the keratinases were studied and compared to those of some known commercial proteases. The profile of the oxidized insulin B-chain digestion revealed that both keratinases, like proteinase K but not subtilisin, trypsin, or elastase, possess broad cleavage specificity with a preference for aromatic and nonpolar amino acid residues at the P-1 position. Kinetic studies were performed on a synthetic substrate, succinyl-Ala-Ala-Pro-Phe-p-nitroanilide. The keratinase of P. marquandii exhibited the lowest K(m) among microbial keratinases reported in the literature, and its catalytic efficiency was high in comparison to that of D. microsporus keratinase and proteinase K. All three keratinolytic enzymes, the keratinases of P. marquandii and D. microsporus as well as proteinase K, were significantly more active on keratin than subtilisin, trypsin, elastase, chymotrypsin, or collagenase

Design of a single-chain polypeptide tetrahedron assembled from coiled-coil segments.

Protein structures evolved through a complex interplay of cooperative interactions, and it is still very challenging to design new protein folds de novo. Here we present a strategy to design self-assembling polypeptide nanostructured polyhedra based on modularization using orthogonal dimerizing segments. We designed and experimentally demonstrated the formation of the tetrahedron that self-assembles from a single polypeptide chain comprising 12 concatenated coiled coil-forming segments separated by flexible peptide hinges. The path of the polypeptide chain is guided by a defined order of segments that traverse each of the six edges of the tetrahedron exactly twice, forming coiled-coil dimers with their corresponding partners. The coincidence of the polypeptide termini in the same vertex is demonstrated by reconstituting a split fluorescent protein in the polypeptide with the correct tetrahedral topology. Polypeptides with a deleted or scrambled segment order fail to self-assemble correctly. This design platform provides a foundation for constructing new topological polypeptide folds based on the set of orthogonal interacting polypeptide segments

Using low-cost GPS receivers for determining field position of mechanized irrigation systems

ABSTRACT. As the accuracy of GPS receivers improves and the costs decrease, more applications for GPS become feasible. One such application is reporting center-pivot and lateral-move field position. Accurate knowledge of center-pivot or lateral-move position in real time is critical for site-specific irrigation. On center pivots, a traditional resolver can only report the location of the first interior tower while a GPS receiver can more precisely show the location of the end of the pivot. This advantage over traditional resolvers becomes more pronounced with longer center pivots. Lateral-move systems do not have a readily available mechanism for reporting their position as they travel over the field. GPS is potentially an ideal method for position and alignment reporting on lateral-moves. The resolver on a three tower center pivot was tested using a survey grade, sub-meter-accuracy GPS receiver. The resolver-reported angular position had errors up to ±5 degrees. Fitting a sine curve and subtracting the modeled errors from the reported measurements corrected these errors to plus or minus one degree. A low-cost GPS receiver was tested in a stationary location on the same center pivot to determine its fitness for reporting field position for mechanized or self-propelled irrigation systems. This low-cost receiver was accurate to within 2.1 m 95 % of the time. However, the remaining 5 % of points showed errors up to 6.6 m. Outlying errors this large can present problems for precision or site-specific irrigation. Suggestions are offered for mitigating these errors

Design of coiled-coil protein-origami cages that self-assemble in vitro and in vivo

Polypeptides and polynucleotides are natural programmable biopolymers that can self-assemble into complex tertiary structures. We describe a system analogous to designed DNA nanostructures in which protein coiled-coil (CC) dimers serve as building blocks for modular de novo design of polyhedral protein cages that efficiently self-assemble in vitro and in vivo. We produced and characterized gt 20 single-chain protein cages in three shapes-tetrahedron, four-sided pyramid, and triangular prism-with the largest containing gt 700 amino-acid residues and measuring 11 nm in diameter. Their stability and folding kinetics were similar to those of natural proteins. Solution small-angle X-ray scattering (SAXS), electron microscopy (EM), and biophysical analysis confirmed agreement of the expressed structures with the designs. We also demonstrated self-assembly of a tetrahedral structure in bacteria, mammalian cells, and mice without evidence of inflammation. A semi-automated computational design platform and a toolbox of CC building modules are provided to enable the design of protein cages in any polyhedral shape.Supplementary material: [http://cherry.chem.bg.ac.rs/handle/123456789/3212

Free Thiol Group of MD-2 as the Target for Inhibition of the Lipopolysaccharide-induced Cell Activation*

MD-2 is a part of the Toll-like 4 signaling complex with an indispensable role in activation of the lipopolysaccharide (LPS) signaling pathway and thus a suitable target for the therapeutic inhibition of TLR4 signaling. Elucidation of MD-2 structure provides a foundation for rational design of inhibitors that bind to MD-2 and inhibit LPS signaling. Since the hydrophobic binding pocket of MD-2 provides little specificity for inhibitors, we have investigated targeting the solvent-accessible cysteine residue within the hydrophobic binding pocket of MD-2. Compounds with affinity for the hydrophobic pocket that contain a thiol-reactive group, which mediates covalent bond formation with the free cysteine residue of MD-2, were tested. Fluorescent compounds 2-(4′-(iodoacetamido)anilino)naphthalene-6-sulfonic acid and N-pyrene maleimide formed a covalent bond with MD-2 through Cys133 and inhibited LPS signaling. Cell activation was also inhibited by thiol-reactive compounds JTT-705 originally targeted against cholesterol ester transfer protein and antirheumatic compound auranofin. Oral intake of JTT-705 significantly inhibited endotoxin-triggered tumor necrosis factor α production in mice. The thiol group of MD-2 also represents the target of environmental or endogenous thiol-reactive compounds that are produced in inflammation

CC-protein-origami

Dataset includes:<p><strong>Topologies-circular-permutations-TCO.xlsx</strong> – List of topologies and circular permutations in file</p><p><strong>CC-protein-origami.fasta</strong> – List of all the design sequences in fasta format.</p><p><strong>all-atom-models.zip</strong> – representative models generated by CoCoPOD, including the models with best fit to SAXS data</p><p><strong>SAXS-data.zip</strong> – SAXS scattering curves of constructs presented in the main article. electron-reconstruction – negative stain density reconstructions.</p><p><b>electron-reconstruction.zip</b> - contains the reconstituted electron microscopy densities in mrc format.<br></p><p><b>Supplementary Source Code.zip </b>– contains the source code of CoCoPOD and all needed files for installation. <b> </b><strong> </strong><br></p><p><br></p