NMR experiments were performed at 25°C on a Bruker Avance III 600 MHz instrument equipped with a TBI probe (Virginia Tech) and on a cold probe-equipped Bruker Avance III 800 MHz instrument (University of Virginia). All NMR samples contained 300 μM of the Phafin2 PH domain in NMR buffer. The NMR protein sample was of high quality as assessed by a series of 1H, 15N-HSQC spectra (Fig. 1a ). Sequential assignments of the protein backbone 1H, 15N, 13C NMR resonances were obtained from two-dimensional 1H, 15N HSQC, as well as three-dimensional CBCA(CO)NH, HNCACB, HNCACO, and HNCO experiments (Grzesiek et al., 1993 ; Muhandiram and Kay, 1994 ). All three-dimensional spectra were 10% nonuniformly sampled. The Poisson Gap method was used to construct sampling schedules (Hyberts et al., 2010 (link)). The NMR data were processed with NMRPipe (Delaglio et al., 1995 (link)) using SMILE (Ying et al., 2017 (link)) for the reconstruction of nonuniformly sampled data. NMRFAM-SPARKY (Lee et al., 2015 (link)) was used for data visualization and sequential resonance assignment. Data processing and analysis was carried out using the NMRbox platform (Maciejewski et al., 2017 (link)).
Tbi probe
Manufactured by Bruker
The TBI probe is a specialized piece of laboratory equipment designed for nuclear magnetic resonance (NMR) spectroscopy. It provides a platform for sample analysis, allowing researchers to obtain detailed information about the chemical and structural properties of a wide range of materials and substances.
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2 protocols using tbi probe
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NMR Characterization of Phafin2 PH Domain
2
NMR Analysis of C. pseudotuberculosis Metabolome
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For this NMR analysis and subsequent metabolomics, 3 distinct groups were studied. In all groups, C. pseudotuberculosis was grown in 10 mL of BHI media. For group 1, the bacterial cultures were grown for 24 h at 200 rpm and 32 °C. To the culture media of groups 2 and 3, 10 mL of ampicillin (0.25 μg mL−1) and 10 mL of AgNPs (20 μg mL−1) were added, respectively, after 16 h of agitation. Then, the cultures were grown for another 8 h, reaching turbidity at 600 nm of 0.8.
By adding ethanol, in the ratio 1 : 1 (v/v) to the bacterial cultures, the growth of C. pseudotuberculosis was quenched. Then, the suspensions were centrifuged for 10 min at 7000 × g at 4 °C, and the cells were suspended in 30 mL of ice-cold PBS (20 mmol L−1, pH = 7.2, 0 °C); this step was repeated twice. The cells were lysed by ultrasonication for 10 s (Ultrasonique). The obtained biological material was centrifuged at 17 000 × g during 2 min, then the supernatant was kept, cell residues were washed, and the supernatants were combined and lyophilized.
Samples for NMR were prepared by dissolving 15 mg of the biological material into 500 μL of deuterium oxide and spectra were acquired on a Bruker Avance III 600 MHz spectrometer with TBI – probe at 25 °C. 1H NMR spectra were acquired as described in analysis section.
By adding ethanol, in the ratio 1 : 1 (v/v) to the bacterial cultures, the growth of C. pseudotuberculosis was quenched. Then, the suspensions were centrifuged for 10 min at 7000 × g at 4 °C, and the cells were suspended in 30 mL of ice-cold PBS (20 mmol L−1, pH = 7.2, 0 °C); this step was repeated twice. The cells were lysed by ultrasonication for 10 s (Ultrasonique). The obtained biological material was centrifuged at 17 000 × g during 2 min, then the supernatant was kept, cell residues were washed, and the supernatants were combined and lyophilized.
Samples for NMR were prepared by dissolving 15 mg of the biological material into 500 μL of deuterium oxide and spectra were acquired on a Bruker Avance III 600 MHz spectrometer with TBI – probe at 25 °C. 1H NMR spectra were acquired as described in analysis section.
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