Protein samples for NMR were buffer exchanged into 25 mM sodium phosphate buffer, 100 mM NaCl, 10% v/v D2O, pH 6.8 in an Amicon Ultra concentrator (EMD Millipore, Billerica, MA) to a final volume of 350 µL. All final protein concentrations were ~35 µM.19F 1D NMR spectra with 1H composite decoupling during acquisition were recorded on a 600 MHz Bruker AVANCE spectrometer, equipped with a CP TXO F/C-H-D triple-resonance z-axis gradient cryoprobe (Bruker Biospin, Billerica, MA). Spectra for the inhibitor-free proteins as well as samples containing NVP, EFV, ETR, and RPV at 1:1 and 1:5 RT: NNRTI inhibitor ratios were recorded using Topspin 3.1 (Bruker) and analyzed with MestReNova (Escondido, CA). Prior to Fourier transformation, the time-domain free-induction decays were apodized with an exponential function, using a line broadening factor of 30 Hz. Chemical shifts and linewidths were calculated using the peak deconvolution feature in MestReNova. An upper limit of uncertainty for the linewidths was qualitatively estimated by assuming that the fit error of each peak is associated with the linewidth error.
Mestrenova
Manufactured by Bruker
Sourced in Germany
MestReNova is a software package for processing and analyzing nuclear magnetic resonance (NMR) data. It provides tools for tasks such as peak assignment, integration, and data visualization. The software supports a range of NMR experiment types and can be used with data from various NMR spectrometers.
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Lab products found in correlation
300 ultrashield, by Bruker (2 mentions)
Topspin 3, by Bruker (2 mentions)
Amicon ultra concentrator, by Merck Group (1 mentions)
Cp txo f c h d triple resonance z axis gradient cryoprobe, by Bruker (1 mentions)
Avance spectrometer, by Bruker (1 mentions)
Topspin version 3, by Bruker (1 mentions)
700 mhz spectrometer, by Bruker (1 mentions)
Aviii 700 mhz nmr spectrometer, by Bruker (1 mentions)
Match nmr tubes, by Bruker (1 mentions)
Topspin, by Bruker (1 mentions)
Toluene, by Thermo Fisher Scientific (1 mentions)
Ethyl acetate, by Thermo Fisher Scientific (1 mentions)
Alpha platinum atr, by Bruker (1 mentions)
Acs grade methanol, by Thermo Fisher Scientific (1 mentions)
Anhydrous dmf, by Thermo Fisher Scientific (1 mentions)
Dimethyl sulfoxide (dmso), by Thermo Fisher Scientific (1 mentions)
Avance 500 mhz spectrometer, by Bruker (1 mentions)
Avance 3 500 mhz, by Bruker (1 mentions)
7 protocols using mestrenova
1
NMR Analysis of NNRTI Inhibitor Binding
2
NMR Spectroscopy of G-Quadruplex RNA
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The one-dimensional 1H NMR spectra were mostly recorded at 308 K on Bruker 700-MHz spectrometers equipped with a regular probe. RNA samples were dissolved in 5 mM Potassium Cacodylate (Kcaco) (pH 6.5), 50 mM KCl, and 10% D2O at a final concentration of 300 μM. The NMR data were processed with Bruker TopSpin Version 3.2 and MestReNova. Formation of the G4 structure was demonstrated by the presence of an imino proton peak in the 10–11.5 ppm region of the 1H NMR spectrum, which is highly characteristic of the Hoogsteen hydrogen bonds of G4 (42 (link)).
3
NMR Characterization of R132H IDH1
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1H NMR assays were conducted using a Bruker AVIII 700 MHz NMR spectrometer equipped with a 5-mm inverse triple-resonance-inverse (TCI) cryoprobe at 298 K. NMR spectra were processed using MestReNova (version 1.10) and TopSpin (version 3.6.1). R132H IDH1 assays were performed in buffer (50 mM Tris-d11, pH 7.5, 10%v/v D2O, 10 mM MgCl2, 150 mM NaCl) using a water suppression pulse sequence (32 scans with a 2 s relaxation delay per time point). Conditions of the R132H IDH1-catalyzed reduction of 2OG derivatives (180 μL total reaction volume in 3 mm diameter MATCH NMR tubes): 0.5 μM R132H IDH1, 1.5 mM 2OG/2OG derivatives, 1.5 mM NADPH.
Binding studies were performed at 298K in buffer (50 mM Tris-d11, pH 7.5, 10%v/v D2O, 10 mM CaCl2) using CPMG NMR spectroscopy (56 ) and a water suppression pulse sequence. Concentrated R132H IDH1 (1.4 mM stock) was titrated (in 2.86 μL increments) into a mixture (160 μL total volume) containing a 2OG derivative (50 μM) in 3 mm diameter MATCH NMR tubes (Bruker).
Binding studies were performed at 298K in buffer (50 mM Tris-d11, pH 7.5, 10%v/v D2O, 10 mM CaCl2) using CPMG NMR spectroscopy (56 ) and a water suppression pulse sequence. Concentrated R132H IDH1 (1.4 mM stock) was titrated (in 2.86 μL increments) into a mixture (160 μL total volume) containing a 2OG derivative (50 μM) in 3 mm diameter MATCH NMR tubes (Bruker).
4
NMR Spectra Acquisition and Processing
5
Synthesis and Characterization of ITZ Analogues
6
NMR Spectroscopy of RNA Structures
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For spectra recorded in 90% H2O/10% D2O water signal was suppressed using the 3–9–19 WATERGATE pulse sequence or excitation sculpting with gradient pulse. The data were processed with TopSpin 3.0 (Bruker BioSpin Gmbh) software and analyzed with MestReNova software. For 1D NMR measurement, RNA samples of 2 mM concentration were dissolved in 150 µl of designed solution containing 10% D2O, 150 mM KCl and 10 mM Tris-HCl buffer (pH 7.0). The 2D NMR spectrum in 90% H2O/10% D2O was collected from 360 scans with 150 ms mixing time at 23 °C. On average, 2048 complex points and 512 FIDs were collected within the spectral width of 14097 Hz. The sample solutions were as follows: 3 mM RNA were dissolved in 150 µl of designed solution containing 10% D2O, 150 mM KCl and 10 mM Tris-HCl buffer (pH 7.0). Samples were prepared by heating the oligonucleotides at 85 °C for 3 min and gradually cooling to room temperature.
7
NMR Spectroscopy of Samples in D2O
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NMR spectra were recorded at 27 °C (300 K) on an Avance III 500 MHz Bruker spectrometer equipped with a 5 mm TBI broadband probe or a 300 Ultrashield from Bruker (Billerica, MA, USA). Data were processed with the software Mestrenova (Bruker GmbH, Karlsruhe, Germany). Samples were prepared at the desired concentration in D2O.
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