A lanthanide-binding tag site-specifically attached to a protein presents a tool to probe the protein by multiple spectroscopic techniques, including nuclear magnetic resonance, electron paramagnetic resonance and time-resolved luminescence spectroscopy. Here a new stable chiral Ln III tag, referred to as C12, is presented for spontaneous and quantitative reaction with a cysteine residue to generate a stable thioether bond. The synthetic protocol of the tag is relatively straightforward, and the tag is stable for storage and shipping. It displays greatly enhanced reactivity towards selenocysteine, opening a route towards selective tagging of selenocysteine in proteins containing cysteine residues.Loaded with Tb III or Tm III ions, the C12 tag readily generates pseudocontact shifts (PCS) in protein NMR spectra. It produces a relatively rigid tether between lanthanide and protein, which is beneficial for interpretation of the PCSs by single magnetic susceptibility anisotropy tensors, and it is suitable for measuring distance distributions in double electron-electron resonance experiments. Upon reaction with cysteine or other thiol compounds, the Tb III complex exhibits a 100-fold enhancement in luminescence quantum yield, affording a highly sensitive turn-on luminescence probe for time-resolved FRET assays and enzyme reaction monitoring.
A mutant aminoacyl-tRNA synthetase identified by a library selection system affords site-specific incorporation of 7-fluoro-L-tryptophan in response to an amber stop codon. The enzyme allows the production of proteins with a single hydrogen atom replaced by a fluorine atom as a sensitive nuclear magnetic resonance (NMR) probe. The substitution of a single hydrogen atom by another element that is as closely similar in size and hydrophobicity as possible minimizes possible perturbations in the structure, stability, and solubility of the protein. The fluorine atom enables site-selective monitoring of the protein response to ligand binding by 19 F NMR spectroscopy, as demonstrated with the Zika virus NS2B-NS3 protease. KEYWORDS: fluoro-tryptophan, 19 F NMR spectroscopy, ligand binding, genetic encoding, noncanonical amino acids, pyrrolysyl-tRNA synthetase 19 F NMR spectroscopy of proteins labeled with fluorine atoms is becoming increasingly popular for studying protein structure, conformational changes, and protein−ligand interactions. 1−3 19
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...
Efficient syntheses of fluorinated leucines, valines and alanines are described. The synthetic routes provide expedient access to various 13C/15N/D isotopologues requiring solely readily available and inexpensive isotope containing reagents such...
Abstract. The metallo-β-lactamase IMP-1 features a flexible loop near the active site that assumes different conformations in single crystal structures, which may assist in substrate binding and enzymatic activity. To probe the position of this loop, we labelled the tryptophan residues of IMP-1 with 7-13C-indole and the protein with lanthanoid tags at three different sites. The magnetic susceptibility anisotropy (Δχ) tensors were determined by measuring pseudocontact shifts (PCS) of backbone amide protons. The Δχ tensors were subsequently used to identify the atomic coordinates of the tryptophan side chains in the protein. The PCSs were sufficient to determine the location of Trp28, which is located in the active site loop targeted by our experiments, with high accuracy. Its average atomic coordinates showed barely significant changes in response to the inhibitor captopril. It was found that localisation spaces could be defined with better accuracy by including only the PCSs of a single paramagnetic lanthanoid ion for each tag and tagging site. The effect was attributed to the shallow angle with which PCS isosurfaces tend to intersect if generated by tags and tagging sites that are identical except for the paramagnetic lanthanoid ion.
The selenol group of selenocysteine is much more nucleophilic than the thiol group of cysteine. Selenocysteine residues in proteins thus offer reactive points for rapid post-translational modification. Herein, we show that selenoproteins can be expressed in high yield and purity by cell-free protein synthesis by global substitution of cysteine by selenocysteine. Complete alkylation of solvent-exposed selenocysteine residues was achieved in 10 minutes with 4-chloromethylene dipicolinic acid (4Cl-MDPA) under conditions that left cysteine residues unchanged even after overnight incubation. Gd III À Gd III distances measured by double electron-electron resonance (DEER) experiments of maltose binding protein (MBP) containing two selenocysteine residues tagged with 4Cl-MDPA-Gd III were indistinguishable from Gd III À Gd III distances measured of MBP containing cysteine reacted with 4Br-MDPA tags.
Abstract. The metallo-β-lactamase IMP-1 features a flexible loop near the active site that assumes different conformations in single crystal
structures, which may assist in substrate binding and enzymatic activity. To
probe the position of this loop, we labelled the tryptophan residues of
IMP-1 with 7-13C-indole and the protein with lanthanoid tags at three
different sites. The magnetic susceptibility anisotropy (Δχ)
tensors were determined by measuring pseudocontact shifts (PCSs) of backbone amide protons. The Δχ tensors were subsequently used to
identify the atomic coordinates of the tryptophan side chains in the
protein. The PCSs were sufficient to determine the location of Trp28, which
is in the active site loop targeted by our experiments, with high accuracy. Its average atomic coordinates showed barely significant changes in response to the inhibitor captopril. It was found that localisation spaces could be defined with better accuracy by including only the PCSs of a single paramagnetic lanthanoid ion for each tag and tagging site. The effect
was attributed to the shallow angle with which PCS isosurfaces tend to
intersect if generated by tags and tagging sites that are identical except
for the paramagnetic lanthanoid ion.
Purpose
Use of highly dye doped nano composite for organic pollutant degradation.
Design/methodology/approach
One-pot synthesis of titanium nano-particles were carried out in the presence of N719 dye.
Findings
High dye doping and exceptional dye degradation efficiency was observed. Within 25 min, 99 per cent of methylene blue was removed from waste water.
Originality/value
A novel one-pot synthesis of the composite was introduced.
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