NMR data were collected on Bruker Advance Neo 600- and 800-MHz spectrometers equipped with TCI HCN Z-gradient cryoprobes at 298 K. NMR measurements of PTP1B were recorded using (2H,15N)-labeled protein at a final concentration of 0.2 mM in NMR buffer and 90% H2O/10% D2O. Data were processed using TopSpin 4.05 (Bruker) and analyzed using CcpNmr (47 (link)). The sequence-specific backbone assignments of PTP1BL204A were achieved using 3D triple resonance experiments including 2D [1H,15N] TROSY, 3D TROSY-HNCA, 3D TROSY-HN(CO)CA, 3D TROSY-HN(CO)CACB, and 3D TROSY-HNCACB. All NMR assignment data were processed using TopSpin 4.05 (Bruker) and analyzed using CARA (computer-aided resonance assignment).
Topspin 4
Manufactured by Bruker
Sourced in Germany, United States, France, Canada, United Kingdom, Australia, Spain, Switzerland, Italy
Topspin 4.0.6 is a software package developed by Bruker for the acquisition, processing, and analysis of nuclear magnetic resonance (NMR) data. It provides a comprehensive suite of tools and features for managing NMR experiments and data.
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Lab products found in correlation
Avance 3, by Bruker (16 mentions)
Avance neo, by Bruker (10 mentions)
Avance 3 hd, by Bruker (7 mentions)
Avance spectrometer, by Bruker (6 mentions)
Topspin 3, by Bruker (6 mentions)
Avance 3 600, by Bruker (5 mentions)
Avance 3 hd spectrometer, by Bruker (4 mentions)
Avance neo spectrometer, by Bruker (4 mentions)
Avance 3 spectrometer, by Bruker (4 mentions)
Avance 3 600 mhz, by Bruker (4 mentions)
5 mm bbo probe, by Bruker (4 mentions)
Nmr suite 8, by Chenomx (3 mentions)
Avance neo 500, by Bruker (3 mentions)
Avance nmr spectrometer, by Bruker (3 mentions)
Nmrpipe, by Bruker (3 mentions)
Avance, by Bruker (3 mentions)
Bbiorefcode 2 0 0, by Bruker (3 mentions)
Avance 3 hd 500 nmr spectrometer, by Bruker (3 mentions)
Drx 800 mhz spectrometer, by Bruker (3 mentions)
Trimethylsilyl propionic acid, by Merck Group (3 mentions)
282 protocols using topspin 4
1
NMR Characterization of PTP1B Mutant
2
NMR Analysis of A2A Adenosine Receptor
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All NMR data were processed and analyzed in Topspin 4.0.8 (Bruker Biospin). All 1-dimensional 19F-NMR data were processed identically. The data were zero-filled to 64k points and multiplied by an exponential window function with 40 Hz line broadening prior to Fourier transformation. 19F spectra were referenced to the signal from trifluoroacetic acid (TFA) at −75.8 ppm, which was set to 0 ppm. Deconvolution of the 19F-NMR data followed previously published procedures and was done with MestreNova version 14.1.1-24571 (Mestrelab Research). For each spectrum, the between the experiment data and sum of the deconvoluted signals was assessed to check the quality of deconvolution. The relative population of the different A2AAR conformational states were calculated as a ratio of integrated area of the each deconvoluted peak to the total integral of all the signals from 6 ppm to 15.5 ppm.
For 31P NMR experiments prior to Fourier transformation, 31P data were zero-filled to 64k points and multiplied by an exponential window function with 50 Hz line broadening. All 31P NMR data were analyzed identically in Topspin 4.0.8 (Bruker Biospin).
For 31P NMR experiments prior to Fourier transformation, 31P data were zero-filled to 64k points and multiplied by an exponential window function with 50 Hz line broadening. All 31P NMR data were analyzed identically in Topspin 4.0.8 (Bruker Biospin).
3
Diffusion Coefficient Measurement of Side-Chain Protons
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Diffusion experiments
were recorded for 1H nuclei using 2D sequence for bipolar
gradient pulse with stimulated echo and longitudinal encoding–decoding
(Bruker pulse sequence ledbpgp2s).45 (link) Gradient strength values were taken
in range of 2–98% in 16 steps. Gradient length was set to 1.5
ms and diffusion time was set to be 120 ms. 32 transients were obtained
for each experiment at time points of 12, 36, and 84 h. All experiments
were recorded at 58 °C.
Processing of the diffusion data
was performed using Bruker Topspin 4.0.7 and the diffusion coefficients
in m2 s–1 were calculated for each of
the side-chain proton peaks using dynamic center suite in Bruker Topspin
4.0.7 after curve fitting using the following equation
were recorded for 1H nuclei using 2D sequence for bipolar
gradient pulse with stimulated echo and longitudinal encoding–decoding
(Bruker pulse sequence ledbpgp2s).45 (link) Gradient strength values were taken
in range of 2–98% in 16 steps. Gradient length was set to 1.5
ms and diffusion time was set to be 120 ms. 32 transients were obtained
for each experiment at time points of 12, 36, and 84 h. All experiments
were recorded at 58 °C.
Processing of the diffusion data
was performed using Bruker Topspin 4.0.7 and the diffusion coefficients
in m2 s–1 were calculated for each of
the side-chain proton peaks using dynamic center suite in Bruker Topspin
4.0.7 after curve fitting using the following equation
4
NMR Characterization of Bioreactor Fermentation
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To confirm the formation of propionate and other SCCA in bioreactor fermentations, we conducted NMR spectrometry in addition to HPLC‐RI. NMR was performed as previously described (Huertas‐Díaz et al., 2023 (link)). Briefly, 200 μL of sodium phosphate buffer (150 mM, pH 7.5) containing 5 mM trimethylsilylpropanoic acid (TSP) as internal chemical shift reference and 30% D2O was added to 400 μL of fermentate collected from the bioreactors. The mixture was transferred to a NMR tube (4″ Tubes 5 mm, Bruker Biospin), and NMR spectroscopy was performed using a Bruker Neo‐IVDR 600 NMR spectrometer, operating at 1H frequency of 600.03 MHz and equipped with a 5 mm 1H BBI probe (Bruker BioSpin). All 1H spectra were referenced to the TSP signal at 0 ppm. Spectra were manually phase and baseline corrected using Topspin 4.09 (Bruker Biospin) and signals were assigned using Chenomx NMR Suite 8.6 (Chenomx Inc), and the Human Metabolome Database (Wishart et al., 2022 (link)). To confirm the identification, we spiked samples with 1 μL of succinate (500 mM).
5
Characterization of Novel Energetic Compounds
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1H and 13C NMR spectra were tested using Bruker Avance NEO 400 MHz spectrometer (400 and 100 MHz, respectively) in d-DMSO. Chemical shifts are reported as δ values relative to internal standard d-DMSO (δ 2.50 for 1H NMR and 39.52 for 13C NMR) using Bruker TopSpin 4.0.9. Infrared spectra (IR) were obtained on a PerkinElmer Spectrum BX FT-IR instrument equipped with an ATR unit at 25 °C using an Omnic software. Elemental analyses of C/H/N were investigated on a Vario EL III Analyzer. The onset decomposition temperature was measured using a TA Instruments discovery DSC25 differential scanning calorimeter at a heating rate of 5 °C min−1 under dry nitrogen atmosphere. Densities were determined at room temperature by a Micromeritics AccuPyc 1345 gas pycnometer. Impact and friction sensitivities were tested by a BAM fallhammer and friction tester. X-ray diffractions of all single crystals were carried out on a Bruker D8 VENTURE diffractometer using Mo-Kα radiation (λ = 0.71073 Å). The crystal structures were produced employing Mercury 2021.1.0 software and XP. All reagents used in the experiment were purchased from Aladdin manufacturers.
6
Quantitative NMR Analysis of Plasma-Treated Samples
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1H-nuclear magnetic resonance (1H-NMR) spectroscopy was performed 5 h after gas plasma treatment, as described previously [22 (link)]. In brief, 400 µL of the sample was mixed with 200 µL of sodium hydrogen phosphate buffer (0.2 mol/L; pH 7.0), made up of 50% D2O, including 1 mmol/L of 3-trimethylsilyl-[2,2,3,3-D4]-1-propanoic acid, for 1H-NMR analysis. An AVANCE-II 600 NMR spectrometer (Bruker, Bremen, Germany) was operated by TOPSPIN 3.2 software. Qualitative and quantitative data analyses were conducted using TOPSPIN 4.0.9 (Bruker Biospin) and Chenomx NMR Suite v9 (Chenomx, Edmonton/Alberta, Canada).
7
NMR Spectrum Processing and Calibration
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NMR spectra were processed using TopSpin 4.0.9 (Bruker BioSpin , Rheinstetten, Germany). The free induction decays (FIDs) were Fourier-transformed with an exponential function with line-broadening factor of 0.3 Hz. All 1H NMR spectra were calibrated to the TSP-d4 signal at 0.00 ppm and processed by automatic zero order phase correction (apk0) and automatic baseline correction (absn).
8
NMR Analysis of Peptide-TssK Interaction
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All NMR experiments were recorded at 300 K on a Bruker Avance-II 600 MHz spectrometer equipped with a cryoprobe at the IMM (Institut de Microbiologie de la Mediterranée) NMR platform. The 450-μl peptide sample tube was prepared at 1.8 mM in KPO4 buffer (50 mM KPO4 [pH 6.9], 150 mM NaCl) complemented with 30 μl D2O. 1D 1H-1H-TOCSY, 1H-NOESY, and 1H-15N HSQC spectra (F2 = 2048; F1 = 128; NS = 384) (NS is the number of scans [accumulation] for the experiment; F2 is the sweep width; F1 is the Fourier number that defines the 2D spectrum dimensions and its digital resolution) were recorded using default pulse sequences as provided by the manufacturer. For the peptide-TssK interaction experiments, 200 μl of a 410 μM stock of TssK was added to the same sample tube (1.2 mM peptide and 140 μM TssK [final concentration]), and all NMR spectra were recorded with the addition of longer 1H-15N HSQC spectrometry (NS = 1,024). All spectra were transformed and the figures were generated using Bruker Topspin 4.0.9.
9
NMR Spectroscopy of Transthyretin
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NMR spectra were obtained at 700 MHz with a Bruker AVANCE NEO (Bruker, Billerica, MA, USA) on 80–125 μM U-[2H,13C,15N] recombinant TTR samples in 10 mM HEPES buffer at pH 6.5 or 7.4 with and without 154 mM NaCl, in a mixture of H2O/D2O 95/5 v/v. A reference 2D [15N, 1H] TROSY spectrum [14 (link)] for apo-TTR was acquired at 25 °C, together with additional spectra at 28, 31, 34, 37 °C, respectively. The spectra at various temperatures were necessary to obtain the assignment at 25 °C from the already known assignment at 37 °C. The 2D [15N, 1H] TROSY spectra were acquired upon addition of small aliquots of stock CaCl2 solutions 0.025–2 M with a maximum volume variation of 1.5%.
The 3D HNCA spectra were acquired at 298 K to check the assignment at the final CaCl2 concentration.
The chemical shift deviation of individual amide pairs, , was defined as [15 (link)]:
Spectra were processed with Topspin 4.0.9 (Bruker Biospin, Billerica, MA, USA) and analyzed in NMRFAM-SPARKY [16 (link)]. The non-linear regression to obtain the dissociation constants was performed with Mathematica 11.
The 3D HNCA spectra were acquired at 298 K to check the assignment at the final CaCl2 concentration.
The chemical shift deviation of individual amide pairs, , was defined as [15 (link)]:
Spectra were processed with Topspin 4.0.9 (Bruker Biospin, Billerica, MA, USA) and analyzed in NMRFAM-SPARKY [16 (link)]. The non-linear regression to obtain the dissociation constants was performed with Mathematica 11.
10
NMR Spectroscopy Workflow Optimization
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All NMR experiments were performed with a Bruker AVANCE NEO 600 MHz spectrometer, equipped with a 5 mm cryogenic probe Prodigy TCI. All NMR spectra were processed with Bruker Topspin 4.0.6 (Bruker BioSpin GmbH, Rheinstetten, Germany) and analyzed using CARA 1.9 [67 ] and NMRFAM-Sparky [68 (link)].
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