NMR samples were prepared by dissolving cyclotides into 80 mM potassium phosphate pH 6.0 in 90% H2O/10% D2O (v/v) to a concentration of approximately 0.5 mM. All 1H-NMR data were recorded on an Avance II 700 MHz spectrometer (Bruker, Billerica, MA, USA) equipped with the TXI cryoprobe. Data were acquired at 298 K, and 2,2-dimethyl-2-silapentane-5-sulfonate (DSS), was used as an internal reference. The carrier frequency was centered on the water signal, and the solvent was suppressed by using WATERGATE pulse sequence. 1H, 1H-TOCSY (spin lock time 80 ms) and 1H, 1H-NOESY (mixing time 150 ms) spectra were collected using 4096 t2 points and 256 t1 of 64 transients. Spectra were processed using Topspin 2.1 (Bruker). Each 2D-data set was apodized by 90°-shifted sinebell-squared in all dimensions, and zero filled to 4096 × 512 points prior to Fourier transformation. Assignments for Hα (H-Cα) and H′ (H-Nα) protons of folded MCo-AT1-7 (Table S1 ) were obtained using standard procedures [34 ,35 ].
Avance 2 700 mhz spectrometer
Manufactured by Bruker
Sourced in United States
The Avance II 700 MHz spectrometer is a high-performance nuclear magnetic resonance (NMR) instrument designed for advanced analytical applications. It features a 700 MHz superconducting magnet and provides precise and accurate measurements of molecular structures and dynamics.
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9 protocols using avance 2 700 mhz spectrometer
1
NMR Characterization of Cyclotides
2
Cyclotide NMR Sample Preparation and Analysis
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NMR samples were prepared by dissolving cyclotides into 80 mM potassium phosphate pH 6.0 in 90% H2O/10% D2O (v/v) to a concentration of approximately 0.5 mM. All 1H-NMR data were recorded on an Avance II 700 MHz spectrometer (Bruker, Billerica, MA, USA) equipped with the TXI cryoprobe. Data were acquired at 298 K, and 2,2-dimethyl-2-silapentane-5- sulfonate (DSS), was used as an internal reference. The carrier frequency was centered on the water signal, and the solvent was suppressed by using WATERGATE pulse sequence. 1H,1H-TOCSY (spin lock time 80 ms) and 1H,1H-NOESY (mixing time 150 ms) spectra were collected using 4096 t2 points and 256 t1 of 64 transients. Spectra were processed using Topspin 2.1 (Bruker). Each 2D-data set was apodized by 90°-shifted sinebell-squared in all dimensions, and zero filled to 4096 × 512 points prior to Fourier transformation. Assignments for Hα (H-Cα) and H’ (H-Nα) protons of folded MCo-AT1-7 (Table S1 ) were obtained using standard procedures [34 ,35 ].
3
Biodiesel Purity Determination by NMR
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The biodiesel or fatty acid methyl ester was purified using column chromatography and then analyzed by 1H NMR and 13C NMR for product confirmation and purity. On a Bruker Avance II 700 MHz spectrometer (Fällanden, Switzerland), the 1H NMR and 13C NMR spectra of the produced biodiesel were recorded using tetramethylsilane (TMS) as an internal reference. Using the integrals for methoxy and methylene groups ( and , respectively in Eq. (3 )), biodiesel conversion was determined by the equation derived by Knothe and Kenar. where C indicates the triglyceride to biodiesel (fatty acid methyl ester) conversion percentage (%), AMe denotes the integration value of the methyl esters, and is the integration value of the methylene protons. Factors 2 and 3 ascribe to the proton number in methylene and the proton number in the methyl ester, respectively. The biodiesel yield was calculated by the equation given by Leung and Guo as given in Eq. (4 ).
4
Structural Characterization of PACT-D3 Mutant
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NMR samples were prepared by dialysis into 20 mM MES pH 6.5, 50 mM NaCl, 5–10 mM TCEP followed by the addition of 10% D2O and 50 μM 4,4-dimethyl-4-silapentane-1-sulfonic acid (DSS). The 2D (1H, 15N) HSQC and EXSY spectra, and 3D experiments for assignment of PACT-D3 L273R, were recorded using a Bruker Avance II 700 MHz spectrometer with a triple-resonance room temperature probe. Spectra for backbone assignment of wild-type (WT) PACT-D3 were recorded on a Bruker 600 MHz Avance II+ spectrometer with triple-resonance cryoprobe, while spectra for side-chain assignment was collected on a Bruker 800 MHz Avance III HD spectrometer with triple-resonance cryoprobe. The 13C filter-edit NOESY experiment was recorded on a 50:50 mixture of [13C,15N]- and [15N]-labelled WT PACT-D3 using a Bruker 700 MHz Avance III HD spectrometer with quadruple-resonance cryoprobe. The high pressure 2D (1H, 15N) HSQC NMR experiments were recorded using a Bruker 800 MHz Avance I spectrometer, equipped with a triple-resonance room temperature probe. The sample was inserted into a ceramic tube (rated to 2.5 kbar) and pressurized with paraffin oil (Sigma) using a high-pressure syringe pump (Daedalus Innovations LLC, PA).
5
Purification and NMR Analysis of Viral Proteases
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WNV and DENV2 proteases were expressed by induction in an M9 medium containing 1 g/L of 15NH4Cl and purified as previously described.26 (link) Briefly, cells were harvested at OD600 0.8 by centrifugation and cell pellets were resuspended in lysis buffer. Protease was purified using Ni2+-NTA resin followed by gel filtration using a Superdex 200 column. Pooled fractions were then buffer exchanged into an NMR buffer (20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 2 mM dithiothreitol and 10% D2O) for structural analysis. Compounds were dissolved in deuterated-DMSO and titrated into a 0.4 mM 15N-labeled protease solution. All NMR spectra were acquired at 298 K on a Bruker Avance II 700 MHz spectrometer (Bruker Corporation, Billerica, MA, USA) equipped with a cryoprobe, and data were processed using NMRPipe and visualized with NMRView.
6
NMR Characterization of Protein-DNA Interactions
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All spectra were recorded at 25°C in 20 mM MES, 150 mM KCl, pH 6.0, 10% D2O, on a Bruker AVANCE II 700 MHz spectrometer with a triple-resonance probe. For spectral assignments using uniformly 13C,15N-labelled protein, HNCO, HN(CA)CO, CBCANH and CBCA(CO)NH experiments were performed with 50% overall non-uniform sampling (NUS) in the indirect dimensions. To aid verification of backbone assignments and assignment of sidechain resonances, 13C-decoupled 15N-1H TOCSY-HSQC and 15N-1H NOESY-HSQC experiments were also performed, with mixing times of 60 and 160 ms, respectively, and 40% overall NUS in the indirect dimensions. Data were reconstructed and processed with mddNMR v2.4 (55 (link)) and NMRPipe (56 (link)), and referenced to 4,4-dimethyl-4-silapentane-1-sulfonic acid (0.1 M) in the sample. Peak lists and assignments are deposited under BMRB entry 25876. For chemical shift perturbation studies DNA and protein were dialysed against the same buffer. Increasing amounts of DNA (7.5 mM) were added to protein (300 μM) to achieve DNA/protein molar ratios of 0.2–19.2 and a series of 1H-15N HSQC spectra were acquired. Spectral assignments and perturbation data analyses were performed in CcpNmr Analysis v2.4.1 (57 (link)). Combined chemical shift differences were given by,
where α = 0.20 for glycine and α = 0.14 for all other residues (58 (link)).
where α = 0.20 for glycine and α = 0.14 for all other residues (58 (link)).
7
Isotopic Labeling of VcDciA Protein
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Production of 15N and 13C labeled VcDciA[1–111] was obtained using the same protocol as described above except that the cellular culture was performed in a minimal medium supplemented with 15N ammonium chloride and 13C glucose (Eurisotop) (30 (link)). NMR experiments were carried out on a Bruker AvanceII-700 MHz spectrometer. For assignment experiments, purified 15N–13C VcDciA[1–111] was concentrated up to 0.9 mM, in the NMR buffer (20 mM phosphate buffer (NH2PO4) pH 5.8, NaCl 50 mM, 2 μM EDTA, 0.2 mM DSS, 0.1 mg/ml NaN3, 10% D2O). Data for assignment of the backbone resonances were collected at 293 K using standard 1H-15N HSQC, 15N-edited NOESY-HSQC, TOCSY-HSQC, HBHA(CO)NH, HN(CA)N, HNCO, HNCA, HN(CO)CA, HN(CA)CO and CBCA(CO)NH experiments. Proton chemical shifts (in ppm) were referenced relative to internal DSS and 15N and 13C references were set indirectly relative to DSS using frequency ratios (31 (link)). NMR data were processed using Topspin (Bruker) and analyzed using SPARKY 3 (T.D. Goddard and D.G. Kneller, UCSF).
8
Circular Dichroism and NMR Spectroscopy
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CD measurements were performed using a Jasko J-815 CD spectrometer equipped with a Peltier temperature controller and single cuvette holder. All 1D and 2D solution NMR spectra were recorded on a Bruker AVANCE II 700 MHz spectrometer equipped with a 5-mm inverse triple resonance probe (see SI for details).
9
Nicastrin Backbone Relaxation Dynamics
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R1, R2 and 1H-15N heteronuclear NOE (hetNOE) experiments50 (link) were measured at 313 K using a 15N-labeled nicastrin on a Bruker Avance II 700 MHz spectrometer. For T1 measurement, the relaxation delays of 50, 80, 130, 330, 470, 630, 800, 900, 1000, 1200, 1400, 1600 and 1800 ms were recorded. For T2 measurement, the data were acquired with delays of 17, 34, 51, 68, 85, 102, 119, 136, and 153 ms. The hetNOE values were obtained using two datasets that were collected with and without initial proton saturation for a period of 3 s51 (link).
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