Interaction studies between FMN and OmcA mutants were performed as previously described for OmcA and mutants OmcA_H4, OmcA_H5, OmcA_H6, OmcA_H8, OmcA_H9, and OmcA_H10 [27 (link),29 (link)]. Briefly, 100 μM FMN samples were titrated against increasing amounts of the target mutant protein and 31P-1D-NMR spectra were recorded after each addition [27 (link)]. The NMR experiments, performed at 25 °C, were acquired on a Bruker Avance II 500 MHz NMR spectrometer equipped with a SEX probe. 31P-1D-NMR experiments were collected with proton decoupling and calibrated using phosphate buffer as an internal reference. Data analysis and binding affinities determination were performed as previously described [29 (link)].
Avance 2 500 mhz nmr spectrometer
Manufactured by Bruker
Sourced in Germany
The Avance II 500 MHz NMR spectrometer is a high-performance nuclear magnetic resonance (NMR) instrument designed for analytical and research applications. It operates at a frequency of 500 MHz and is capable of acquiring high-resolution NMR spectra for the identification and characterization of chemical compounds.
Automatically generated - may contain errors
Lab products found in correlation
Avance 2 500 mhz spectrometer, by Bruker (1 mentions)
Hp1200 system, by Agilent Technologies (1 mentions)
Spectrum 100 spectrophotometer, by PerkinElmer (1 mentions)
Uv 1800 spectrophotometer, by PerkinElmer (1 mentions)
Microtof focus mass spectrometer, by Bruker (1 mentions)
P 1030 polarimeter, by Jasco (1 mentions)
Topspin 4, by Bruker (1 mentions)
5 mm tci c n prodigy cryoprobe, by Bruker (1 mentions)
8 protocols using avance 2 500 mhz nmr spectrometer
1
FMN Binding Interactions with OmcA Mutants
2
Investigating OcwA Interactions with Electron Shuttles
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For all of the NMR experiments, OcwA prepared in 20 mM potassium phosphate buffer (pH 7.6) with 100 mM KCl was lyophilized and resuspended in D2O. An excess of sodium dithionite was used to reduce the protein. 1H NMR experiments were performed on a Bruker Avance II 500 MHz spectrometer equipped with a QXI probe for 1H detection and an SEX probe for 31P detection. All NMR data were processed in the TopSpin 3.2 software. 1H NMR spectra were acquired before and after lyophilization to ensure that protein integrity was preserved.
To study the influence of the electron shuttles on OcwA, NMR experiments were performed as previously described (31 (link)) using antraquinone-2,6-disulfonate (AQDS), flavin mononucleotide (FMN), riboflavin (RF), and phenazine methosulfate (PMS). Stock solutions of the different electron shuttles were prepared in 20 mM potassium phosphate buffer (pH 7.6) with 100 mM KCl. 1H NMR spectra performed at 25°C on a Bruker Avance II 500 MHz NMR spectrometer equipped with a TCI cryoprobe for 1H detection were acquired before and after the addition of the electron shuttles (molar ratios 0.5:1, 1:1, and 3:1 of electron shuttle to protein).
For the 31P-NMR binding experiments, samples containing 100 μM FMN prepared in 20 mM phosphate buffer (pH 7.6) with 100 mM KCl were titrated against increasing concentrations of OcwA at 25°C.
To study the influence of the electron shuttles on OcwA, NMR experiments were performed as previously described (31 (link)) using antraquinone-2,6-disulfonate (AQDS), flavin mononucleotide (FMN), riboflavin (RF), and phenazine methosulfate (PMS). Stock solutions of the different electron shuttles were prepared in 20 mM potassium phosphate buffer (pH 7.6) with 100 mM KCl. 1H NMR spectra performed at 25°C on a Bruker Avance II 500 MHz NMR spectrometer equipped with a TCI cryoprobe for 1H detection were acquired before and after the addition of the electron shuttles (molar ratios 0.5:1, 1:1, and 3:1 of electron shuttle to protein).
For the 31P-NMR binding experiments, samples containing 100 μM FMN prepared in 20 mM phosphate buffer (pH 7.6) with 100 mM KCl were titrated against increasing concentrations of OcwA at 25°C.
3
Temperature-dependent NMR Spectroscopy of Redox Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A sample of approximately 300 μM of reduced (as purified) PioC and Rpal_4085 in 50 mM potassium phosphate buffer pH 7.6 with 300 mM KCl with 10% D2O was used for the 1H temperature dependence experiments. The 1H NMR experiments were performed using a Bruker Avance II 500 MHz NMR spectrometer equipped with a 5 mm PAQXI probe. A total of 3584 transients were acquired using the super-WEFT (water-eliminated Fourier transform) pulse sequence (180-τ-90-AQ) with 122 ms of recycle time and τ values of 119 ms to dampen the diamagnetic signals and suppress the solvent.
4
NMR Kinetic Analysis of PdGH110B
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Recombinant PdGH110B (1.3 ml at 1 mg/ml) was buffer-exchanged overnight at 4 °C in 1 liter of 50 mm sodium phosphate buffer (pH 5.8). PdGH110B was then buffer-exchanged into NMR buffer (50 mm sodium phosphate (pH 5.9) in 99.9% D2O) through dilution and reconcentration in an Amicon centrifugal filtration unit with a 10-kDa cutoff. The reaction contained 8.3 mm pNP-α-d -galactopyranoside and 2.9 μm recombinant PdGH110B in NMR buffer. A 10 mm d -galactose control reaction in NMR buffer was also monitored as a standard. The reactions were measured before the addition of PdGH110B and after subsequent incubations at the 5-min, 15-min, 30-min, 1-h, 2-h, and 24-h time points using a Bruker Avance II 500 MHz NMR spectrometer and a 5-mm TXI inverse probe. The data were processed using the MestReNova 10 software package.
5
Spectroscopic Analysis of Organic Compounds
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The specific rotations were measured on a JASCO P-1030 polarimeter. UV and IR spectra were recorded on a Shimadzu UV-1800 spectrophotometer and a PerkinElmer Spectrum 100 spectrophotometer, respectively. NMR spectra were obtained on a Bruker AVANCE II 500 MHz NMR spectrometer in DMSO-d6 supplemented with a trace amount of trifluoroacetic acid using the signals of the residual solvent protons (δH 2.49) and carbon atoms (δC 39.5) as internal standards for compounds 1 –5 , or in CDCl3 using the signals of the residual solvent protons (δH 7.27) and carbon atoms (δC 77.0) as internal standards for other compounds. HRESITOFMS spectra were recorded on a Bruker micrOTOF focus mass spectrometer. An Agilent HP1200 system equipped with a diode array detector was used for analysis and purification.
6
Microwave-Assisted Organic Synthesis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All chemicals were procured from SD Fine Chem Co. Ltd and used in experiments. The melting point has been identified in the Bouchi oil melting point apparatus and has not been corrected. The microwave-assisted reactions were carried out in an HPL 2300 ET domestic microwave oven: power: 300 W; frequency: 2.45 GHz; temperature range: 60–250 °C [29 ]. The purity of the compounds was determined by a single spot on the TLC silica gel G plate. IR spectra were recorded on the Shimadzu IR convergence spectrometer using the KBr pellet method. NMR spectra (1H NMR and 13C NMR) are recorded on the Bruker Avance II 500 MHz NMR spectrometer. Mass spectra were performed on the JEOL GC mass spectrometer. The results in the spectrum head show that the molecular mass of the compounds produced was closer to the molecular mass of the expected compounds [30 ].
7
Characterization of Mutant Cytochrome OmcA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The mutant proteins OmcA_H1, OmcA_H3, OmcA_H4, OmcA_H6, OmcA_H8, OmcA_H9, and OmcA_H10 where the distal histidine ligand of the respective heme has been modified to a methionine, were produced and purified as previously described [29 (link)]. The purity of the proteins was verified by a single band in the SDS-PAGE and by an A408/A280 ratio of above 5 measured by UV-visible spectroscopy. All the proteins were washed with 20 mM phosphate buffer, 100 mM KCl at pH 7.6. This buffer was used for all experiments. The concentration of the proteins was determined by UV-visible spectroscopy using a ε408 nm of 125,000 M−1 cm−1 per heme for the oxidized state of the cytochrome. 1H-1D-NMR spectra were collected for the different mutants on a Bruker Avance II+ 500 MHz NMR spectrometer equipped with a 5 mm TCI C/N prodigy cryoprobe. These experiments were performed at 25 °C.
8
NMR Characterization of Bacterial Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
1H-1D-NMR spectra were collected at 25 °C on an Avance II+ 500 MHz NMR spectrometer (Bruker, Rheinstetten, Germany) equipped with a 5 mm TCI C/N Prodigy Cryo probe. The ImdcA sample was prepared in 20 mM Tris-HCl buffer (pH 9.0) containing 100 mM NaCl and 0.2% SB12 containing 10% 2H2O (99.9 atom%), while the PdcA sample was prepared in 20 mM Tris-HCl buffer (pH 9.0) with 100 mM KCl and 10 mM sodium cholate containing 10% 2H2O (99.9 atom%). For solvent suppression and enhancement of the paramagnetic signals, a SuperWEFT pulse sequence was applied. The NMR spectra were processed and analyzed using Bruker TopSpin 4.0 software.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!