The non-structural 3 protease (NS3pro) is an essential flaviviral enzyme and therefore one of the most promising targets for drug development against West Nile virus (WNV) and dengue infections.
In ...this work, a small-molecule inhibitor of the WNV NS3pro has been identified by automatic fragment-based docking of about 12000 compounds and testing by nuclear magnetic resonance (NMR) spectroscopy of only 22 molecules. Specific binding of the inhibitor into the active site of NS3pro and its binding mode are confirmed by 15N-HSQC NMR spectra. The inhibitory activity is further validated by an enzymatic assay and a tryptophan fluorescence quenching assay.
The inhibitor 4-(carbamimidoylsulfanylmethyl)-2,5-dimethylphenyl-methylsulfanylmethanimidamide has a good ratio of binding affinity versus molecular weight (ligand efficiency of 0.33 kcal/mol per non-hydrogen atom), and thus has good potential as lead compound for further development to combat West Nile virus infections.
Cell-free protein synthesis using eCells allows production of amino acids from inexpensive
13
C-labelled precursors. We show that the metabolic pathway converting pyruvate, glucose and erythrose into ...aromatic amino acids is maintained in eCells. Judicious choice of
13
C-labelled starting material leads to proteins, where the sidechains of aromatic amino acids display
13
C,
1
H-HSQC cross-peaks free of one-bond
13
C–
13
C couplings. Selective
13
C-labelling of tyrosine and phenylalanine residues is achieved simply by using different compositions of the reaction buffers.
Quick and clean: Excellent protein yields in cell‐free protein synthesis can be obtained by using polymerase chain reaction (PCR) amplified DNA templates, provided that the templates are designed for ...cyclization. This eliminates time‐consuming cloning steps and allows one to go from complementary DNA to the protein NMR spectrum in 24 h.
Paramagnetic chemical probes have been used in electron paramagnetic resonance (EPR) and nuclear magnetic resonance (NMR) spectroscopy for more than four decades. Recent years witnessed a great ...increase in the variety of probes for the study of biological macromolecules (proteins, nucleic acids, and oligosaccharides). This Review aims to provide a comprehensive overview of the existing paramagnetic chemical probes, including chemical synthetic approaches, functional properties, and selected applications. Recent developments have seen, in particular, a rapid expansion of the range of lanthanoid probes with anisotropic magnetic susceptibilities for the generation of structural restraints based on residual dipolar couplings and pseudocontact shifts in solution and solid state NMR spectroscopy, mostly for protein studies. Also many new isotropic paramagnetic probes, suitable for NMR measurements of paramagnetic relaxation enhancements, as well as EPR spectroscopic studies (in particular double resonance techniques) have been developed and employed to investigate biological macromolecules. Notwithstanding the large number of reported probes, only few have found broad application and further development of probes for dedicated applications is foreseen.
Paramagnetic metal ions can induce molecular alignment with respect to the magnetic field. This alignment generates residual anisotropic chemical shifts (RACS) due to nonisotropic averaging over the ...molecular orientations. Using a 30 kDa protein−protein complex, the RACS effects are shown to be significant for heteronuclear spins with large chemical shift anisotropies, lanthanide ions with large anisotropic magnetic susceptibility tensors, and measurements at high magnetic field. Therefore, RACS must be taken into account when pseudocontact shifts are measured by comparison of chemical shifts observed between complexes with paramagnetic and diamagnetic lanthanide ions. The results are of particular importance when different pseudocontact shifts measured for the 1HN, 15N, and 13C‘ spins of a peptide group are used to restrain its orientation with respect to the electronic magnetic susceptibility tensor in structure calculations.
Synthesis of indoles labeled with
13
C-
1
H and
13
C-
19
F spin pairs is described. All syntheses utilize inexpensive carbon-
13
C dioxide as the
13
C isotope source. Ruthenium-mediated ring-closing ...metathesis is the key step in construction of the
13
C containing indole carbocycle. Fluorine is introduced
via
electrophilic fluorination at the 7-position and
via
palladium-mediated cross-coupling at the 4-position. Indole and fluoroindoles are viable tryptophan precursors for
in vivo
protein expression. We show that they are viable also in
in vitro
protein synthesis using standard
E. coli
S30 extracts. Incorporation of the synthesized
13
C-
1
H and
13
C-
19
F spin pair labeled tryptophans into proteins enables high-resolution and high-sensitivity nuclear magnetic resonance (NMR) spectroscopy.
Cell-free protein synthesis using
19
F-
13
C and
1
H-
13
C spin pair containing indoles as tryptophan precursors enables site-specific labeling of proteins for
19
F NMR and TROSY NMR studies.
Synthesis of indoles labeled with 13C–1H and 13C–19F spin pairs is described. All syntheses utilize inexpensive carbon–13C dioxide as the 13C isotope source. Ruthenium-mediated ring-closing ...metathesis is the key step in construction of the 13C containing indole carbocycle. Fluorine is introduced via electrophilic fluorination at the 7-position and via palladium-mediated cross-coupling at the 4-position. Indole and fluoroindoles are viable tryptophan precursors for in vivo protein expression. We show that they are viable also in in vitro protein synthesis using standard E. coli S30 extracts. Incorporation of the synthesized 13C–1H and 13C–19F spin pair labeled tryptophans into proteins enables high-resolution and high-sensitivity nuclear magnetic resonance (NMR) spectroscopy.
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•NT* fusion of human rhinovirus 14 3C protease for high-yield expression.•Stable protease for tag removal at low temperature in minutes.•Alternative to tag cleavage with TEV protease.
...The human rhinovirus 14 3C protease (HRV3C protease), in fusion with glutathione S-transferase also referred to as PreScission™ protease, is a cysteine protease of particular interest for affinity tag removal from fusion proteins due to its stringent recognition sequence specificity (LEVLFQ/GX) and superior activity at low temperature. Here we report the expression, purification and use of a fusion construct of HRV3C protease, NT*-HRV3CP, that affords high expression yield in E. coli (over 300 mg/L cell culture), facile single-step purification, high solubility (>10 mg/mL) and excellent storage properties. NT*-HRV3CP cleaves affinity tags at 4 °C in minutes, making it an attractive tool for the production of recombinant proteins for biotechnological, industrial and pharmaceutical applications.
In bacteria, the DnaG primase is responsible for synthesis of short RNA primers used to initiate chain extension by replicative DNA polymerase(s) during chromosomal replication. Among the proteins ...with which
DnaG interacts is the single-stranded DNA-binding protein, SSB. The C-terminal hexapeptide motif of SSB (DDDIPF; SSB-Ct) is highly conserved and is known to engage in essential interactions with many proteins in nucleic acid metabolism, including primase. Here, fragment-based screening by saturation-transfer difference nuclear magnetic resonance (STD-NMR) and surface plasmon resonance assays identified inhibitors of the primase/SSB-Ct interaction. Hits were shown to bind to the SSB-Ct-binding site using
N-¹H HSQC spectra. STD-NMR was used to demonstrate binding of one hit to other SSB-Ct binding partners, confirming the possibility of simultaneous inhibition of multiple protein/SSB interactions. The fragment molecules represent promising scaffolds on which to build to discover new antibacterial compounds.
Apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) is an adaptor protein composed of two homophilic protein−protein interaction domains, a PYRIN domain (PYD) and a ...caspase recruitment domain. PYD-dependent oligomerization of ASC is thought to play a crucial role in formation of a molecular platform, the inflammasome, which activates caspase-1. When expressed in cells, the PYD of ASC was shown to form cytoplasmic filaments through self-association. Over 70 single point mutants were analyzed for filament formation in cells expressing the mutant proteins. The set of mutations comprised every single amino acid residue with a charged side chain (Arg, Lys, Asp, and Glu) and a large hydrophobic side chain (Ile, Leu, Met, Phe, Pro, and Val). Filament formation of the ASC PYD was prevented by mutation of Lys21, Leu25, Lys26, Pro40, Arg41, Asp48, and Asp51 of helices 2, 3, and 4. These data identify a coherent interaction surface, establishing a molecular model of PYD−PYD complexes with an important role for charge−charge interactions.
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