Search

Metagenomics is unearthing the previously hidden world of soil viruses. Many soil viral sequences in metagenomes contain putative auxiliary metabolic genes (AMGs) that are not associated with viral replication. Here, we establish that AMGs on soil viruses actually produce functional, active proteins. We focus on AMGs that potentially encode chitosanase enzymes that metabolize chitin - a common carbon polymer. We express and functionally screen several chitosanase genes identified from environmental metagenomes. One expressed protein showing endo-chitosanase activity (V-Csn) is crystalized and structurally characterized at ultra-high resolution, thus representing the structure of a soil viral AMG product. This structure provides details about the active site, and together with structure models determined using AlphaFold, facilitates understanding of substrate specificity and enzyme mechanism. Our findings support the hypothesis that soil viruses contribute auxiliary functions to their hosts.

Amelogenin constitutes ~90% of the enamel matrix in the secretory stage of amelogenesis, a still poorly understood process that results in the formation of the hardest and most mineralized tissue in vertebrates—enamel. Most biophysical research with amelogenin uses recombinant protein expressed in Escherichia coli. In addition to providing copious amounts of protein, recombinant expression allows 13C‐ and 15N‐labeling for detailed structural studies using NMR spectroscopy. However, native amelogenin is phosphorylated at one position, Ser‐16 in murine amelogenin, and there is mounting evidence that Ser‐16 phosphorylation is important. Using a modified genetic code expansion protocol we have expressed and purified uniformly 13C‐, 15N‐labeled murine amelogenin (pS16M179) with ~95% of the protein being correctly phosphorylated. Homogeneous phosphorylation was achieved using commercially available, enriched, 13C‐, 15N‐labeled media, and protein expression was induced with isopropyl β‐D‐1‐thiogalactopyranoside at 310 K. Phosphoserine incorporation was verified from one‐dimensional 31P NMR spectra, comparison of 1H‐15N HSQC spectra, Phos‐tag SDS PAGE, and mass spectrometry. Phosphorus‐31 NMR spectra for pS16M179 under conditions known to trigger amelogenin self‐assembly into nanospheres confirm nanosphere models with buried N‐termini. Lambda phosphatase treatment of these nanospheres results in the dephosphorylation of pS16M179, confirming that smaller oligomers and monomers with exposed N‐termini are in equilibrium with nanospheres. Such 13C‐, 15N‐labeling of amelogenin with accurately encoded phosphoserine incorporation will accelerate biomineralization research to understand amelogenesis and stimulate the expanded use of genetic code expansion protocols to introduce phosphorylated amino acids into proteins. [ABSTRACT FROM AUTHOR]

Short-chain amphipathic peptides are promising components in the new generation of engineered biomaterials. The model 14-residue leucine-lysine peptide Ac-LKKLLKLLKKLLKL-N[H.sub.2] (LKα) is one such amphipathic peptide. In dilute aqueous solution ( Key words: biomaterials, amphipathic peptide, NMR spectroscopy, circular dichroism spectroscopy, fluorinated alcohols. Resume : Les peptides amphipathiques a chame courte sont des composes tres prometteurs qui font partie de la nouvelle generation de produits issus du genie des biomateriaux. Le peptide modele contenant 14 residus leucine et lysine AcLKKLLKLLKKLLKL-N[H.sub.2] (LKα) est l'un d'entre eux. On a suggere anterieurement, en s'appuyant sur des donnees de spectroscopie DC, qu'en solution aqueuse diluee ( Mots-cles: biomateriaux, peptide amphipathique, spectroscopie RMN, spectroscopie de dichroisme circulaire, alcools fluores., Introduction Peptides play a wide variety of biochemical roles in multicellular organisms, ranging from weapons for antimicrobial destruction (1) to messengers for intercellular communication. (2) Their biological importance is highlighted [...]

The Mycobacterium tuberculosis protein Rv2302 (80 residues; molecular mass of 8.6 kDa) has been characterized using nuclear magnetic resonance (NMR) and circular dichroism (CD) spectroscopy. While the biochemical function of Rv2302 is still unknown, recent microarray analyses show that Rv2302 is upregulated in response to starvation and overexpression of heat shock proteins and, consequently, may play a role in the biochemical processes associated with these events. Rv2302 is a monomer in solution as shown by size exclusion chromatography and NMR spectroscopy. CD spectroscopy suggests that Rv2302 partially unfolds upon heating and that this unfolding is reversible. Using NMR-based methods, the solution structure of Rv2302 was determined. The protein contains a five-strand, antiparallel [beta]-sheet core with one [alpha]-terminal [alpha]-helix (A61 to A75) nestled against its side. Hydrophobic interactions between residues in the [alpha]-helix and [beta]-strands 3 and 4 hold the [alpha]-helix near the [beta]-sheet core. The electrostatic potential on the solvent-accessible surface is primarily negative with the exception of a positive arginine pocket composed of residues R18, R70, and R74. Steady-state {[sup.1]H}-[sup.15]N heteronuclear nuclear Overhauser effects indicate that the protein's core is rigid on the picosecond timescale. The absence of amide cross-peaks for residues G13 to H19 in the [sup.1]H-[sup.15]N heteronuclear single quantum correlation spectrum suggests that this region, a loop between [beta]-strands 1 and 2, undergoes motion on the millisecond to microsecond timescale. Dali searches using the structure closest to the average structure do not identify any high similarities to any other known protein structure, suggesting that the structure of Rv2302 may represent a novel protein fold.

Research has been conducted on the formamidopyrimidine-DNA glycosylase, a protein important for the excision repair of the oxidatively damaged DNA in Escherichia coli. Results indicate that zinc-associated motif is essential for the damaged DNA recognition.

The DNA repair enzyme XPA can bind to DNA even in the absence of a lesion. There may be a cooperative DNA-binding mode between XPA and the largest subunit of replication protein A.

Naturally occurring, pharmacologically active peptides constrained with covalent crosslinks generally have shapes that have evolved to fit precisely into binding pockets on their targets. Such peptides can have excellent pharmaceutical properties, combining the stability and tissue penetration of small-molecule drugs with the specificity of much larger protein therapeutics. The ability to design constrained peptides with precisely specified tertiary structures would enable the design of shape-complementary inhibitors of arbitrary targets. Here we describe the development of computational methods for accurate de novo design of conformationally restricted peptides, and the use of these methods to design 1847 residue, disulfide-crosslinked peptides, a subset of which are heterochiral and/or NC backbone-cyclized. Both genetically encodable and non-canonical peptides are exceptionally stable to thermal and chemical denaturation, and 12 experimentally determined X-ray and NMR structures are nearly identical to the computational design models. The computational design methods and stable scaffolds presented here provide the basis for development of a new generation of peptide-based drugs., Author(s): Gaurav Bhardwaj [1, 2]; Vikram Khipple Mulligan [1, 2]; Christopher D. Bahl [1, 2]; Jason M. Gilmore [1, 2]; Peta J. Harvey [3]; Olivier Cheneval [3]; Garry W. Buchko [...]