In vitro samples were prepared by adding 1 mg of purified SH3 to NMR buffer [50 mM acetic acid/sodium acetate, Hepes, and bis-tris propane (pH 7.8)] prepared in 99.9% D2O. Stated pH values for D2O-containing buffers are pH meter readings and uncorrected for the isotope effect. Methods for acquiring 19F spectra were similar to those used previously [10 (link)]. Spectra were acquired on a Bruker Avance III HD spectrometer with a QCI cryoprobe operating at a Larmor frequency of 470 MHz and running TopSpin Version 3.2. Sodium 2,2-dimethyl-2-silapentane-5-sulfonate (Cambridge Isotope Laboratories) was added to in vitro samples and used to reference the spectra via the Xi factor for 19F. In-cell samples were not referenced because we were concerned with protein stability and only needed the areas under the resonances. Spectra were acquired at 25 °C. The D2O in the sample was used to lock the spectrometer. A sweep width of either 70 ppm or 20 ppm was used, and the number of scans varied from 64 to 128 for in vitro experiments and 128 to 256 for in-cell experiments.
Sodium 2 2 dimethyl 2 silapentane 5 sulfonate
Manufactured by Cambridge Isotopes
Sodium 2,2-dimethyl-2-silapentane-5-sulfonate is a chemical compound used as a reference standard in nuclear magnetic resonance (NMR) spectroscopy. It serves as an internal standard for the calibration and referencing of NMR spectra.
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3 protocols using sodium 2 2 dimethyl 2 silapentane 5 sulfonate
1
NMR Characterization of Purified SH3
2
Alcohol Dehydrogenase Enzyme Assay
3
NMR Characterization of Cyanide-Bound Heme Protein
Check if the same lab product or an alternative is used in the 5 most similar protocols
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A 0.5 mM solution of cyanide-bound
heme in 20 mM Tris, pH 7.4, 50 mM NaCl was prepared as described above.
Purified apo-MhuD and cyanide-bound heme were combined in a 0.9:1
ratio (cyanide-bound heme/apo-MhuD) to produce MhuD–heme–CN.
Residual cyanide-bound heme and apoprotein were removed upon buffer
exchange into 20 mM NaPi, pH 7.4, on a PD-10 column. A 1.5 mM sample
of MhuD–heme–CN in 100% D2O (Cambridge Isotope
Laboratories) was prepared by reducing the sample volume to 600 μL
in Amicon stirred cells (Millipore) followed by lyophilization for
at least 12 h on a VirTis lyophilizer. The resulting powder was dissolved
in 600 μL of D2O.
NMR experiments were conducted
at 11, 25, 35, and 42 °C on a Varian Unity Inova 500 MHz NMR
spectrometer equipped with an inverse triple-resonance probe. 1H NMR experiments utilized a 1.5 s relaxation delay and a
1 s acquisition time. 1H Super water elimination Fourier
transform (Super-WEFT)39 ,40 experiments used a 50 ms τ-delay
and a 50 ms acquisition time. All 1H NMR and 1H Super-WEFT data were referenced to sodium 2,2-dimethyl-2-silapentane-5-sulfonate
(Cambridge Isotope Laboratories) via the residual water peak and processed
using the ACD/Laboratories NMR Processor with 10 Hz exponential line
broadening and zero filling to 65 536 points.
heme in 20 mM Tris, pH 7.4, 50 mM NaCl was prepared as described above.
Purified apo-MhuD and cyanide-bound heme were combined in a 0.9:1
ratio (cyanide-bound heme/apo-MhuD) to produce MhuD–heme–CN.
Residual cyanide-bound heme and apoprotein were removed upon buffer
exchange into 20 mM NaPi, pH 7.4, on a PD-10 column. A 1.5 mM sample
of MhuD–heme–CN in 100% D2O (Cambridge Isotope
Laboratories) was prepared by reducing the sample volume to 600 μL
in Amicon stirred cells (Millipore) followed by lyophilization for
at least 12 h on a VirTis lyophilizer. The resulting powder was dissolved
in 600 μL of D2O.
NMR experiments were conducted
at 11, 25, 35, and 42 °C on a Varian Unity Inova 500 MHz NMR
spectrometer equipped with an inverse triple-resonance probe. 1H NMR experiments utilized a 1.5 s relaxation delay and a
1 s acquisition time. 1H Super water elimination Fourier
transform (Super-WEFT)39 ,40 experiments used a 50 ms τ-delay
and a 50 ms acquisition time. All 1H NMR and 1H Super-WEFT data were referenced to sodium 2,2-dimethyl-2-silapentane-5-sulfonate
(Cambridge Isotope Laboratories) via the residual water peak and processed
using the ACD/Laboratories NMR Processor with 10 Hz exponential line
broadening and zero filling to 65 536 points.
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