The protocol for monitoring the reaction of N1 or N2 with 2,3-butadienoate (allene), 2-butynoate, and propiolate by 1H NMR spectroscopy followed that described elsewhere.25 (link),26 (link) Accordingly, an amount of each compound (4 mg) was added to 100 mM Na2HPO4 buffer (pH ~ 9.2) containing 30 μL of dimethyl sulfoxide-d6 (DMSO-d6) (final total volume of 600 μL). The final pH of the solution was adjusted to 8.0. Subsequently, an aliquot of N1 or N2 was added to the mixture such that the final amount of protein was 0.5 mg. For overnight NMR runs (i.e., 18 h) product formation was determined using a Bruker AVANCE III 500 MHz spectrometer (Billerica, MA). For shorter NMR runs, product formation was monitored every 3 min for 10 intervals on a Varian DirectDrive 600 MHz spectrometer (Palo Alto, CA). The initial spectrum was recorded 3 min after mixing. DMSO-d6 was used as the lock signal and for standardization of the chemical shifts (at 2.49 ppm). The chemical shifts for the products are reported elsewhere.7 (link),26 (link),27 (link) The approximate amount of product in the mixture was determined by integration, as reported elsewhere.26 (link) NMR signals were analyzed using the software program SpinWorks 3.1.6 (Copyright © 2009 Kirk Marat, University of Manitoba).
Directdrive 600 mhz spectrometer
Manufactured by Agilent Technologies
Sourced in United States
The DirectDrive 600 MHz spectrometer is a high-performance analytical instrument designed for laboratory applications. It operates at a frequency of 600 MHz and provides precise data collection and analysis capabilities.
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Lab products found in correlation
Avance 3 500 mhz spectrometer, by Bruker (1 mentions)
Mestrenova 10, by Mestrelab Research (1 mentions)
Inova 500, by Agilent Technologies (1 mentions)
8453 diode array spectrophotometer, by Agilent Technologies (1 mentions)
Lcq electrospray ion trap mass spectrometer, by Thermo Fisher Scientific (1 mentions)
4 protocols using directdrive 600 mhz spectrometer
1
Monitoring Enzyme Reactions by 1H NMR
2
NMR Characterization of Protein-SDS Interactions
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NMR experiments were performed on a Varian Direct Drive 600 MHz spectrometer (California, USA) at 25°C. For each experiment samples were freshly prepared in deuterium oxide at a protein concentration of 100 μM in 5 mM sodium phosphate buffer, 0.05% sodium azide, pH 7.2, containing the desired SDS concentration. Diffusion-ordered spectroscopy (DOSY) spectra were obtained using a gradient length of 3 ms and a diffusion delay of 250 ms. Gradient strength was previously calibrated using the HDO signal in a doped D2O standard. The DOSY data sets were composed by 30 gradient strengths with 128 scans every increment. The DOSY data were processed using MestreNova 10.0 (Mestrelab Research S.L, Spain). Diffusion coefficients were obtained by fitting the intensity decays versus the gradient strength as described elsewhere [46 ]. The hydrodynamic radii were calculated with the Stokes-Einstein equation, Rh = kBT/6πηD, where kB is the Boltzmann constant, T is the absolute temperature, D is the diffusion coefficient and η is the viscosity of D2O at 298 K.
3
Steady-State Kinetic Assays and Protein Characterization
4
NMR Spectroscopy of Protein-Peptide Complexes
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NMR experiments were performed on a Varian Direct Drive 600 MHz spectrometer (Varian, California, USA). The protein samples were labelled with 15 N following a protocol established elsewhere 58 . The protein was dissolved at 0.8 mM in 50 mM sodium phosphate pH 7.4, containing 10% D2O. To prepare a protein-peptide mixture, the lyophilized peptide was dissolved in H20 at a concentration of 1.6 mM and the pH was adjusted to pH 7.4. Then this peptide solution was further lyophilized. Subsequently, the newly lyophilized peptide was dissolved with the protein solution to reach a final concentration of 1.6 mM. 1 H-15 N 2D-HSQC spectra were acquired with 2K points and 32 increments and processed with NMRPipe 59 .
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