For NMR measurements, Fab/Fc fragments and full-length IgG glycoproteins were dissolved in 0.5 mL of 5 mM sodium phosphate buffer [pH 6.0, containing 50 mM NaCl and 10% (v/v) D2O] at a protein concentration of 10 mg/mL. Two-dimensional methyl-transverse relaxation optimized spectroscopy(TROSY) spectral data were acquired at 37 °C using an AVANCE 800 spectrometer equipped with a cryogenic probe (Bruker BioSpin, Fällanden, Switzerland). Assignments for the methionyl methyl resonances of Fc were made based on the previously reported backbone assignments [30 (link)] by analyzing nuclear Overhauser effect (NOE) connectivities observed using AVANCE 800 and AVANCEIII 900 spectrometers equipped with cryogenic probes (Bruker BioSpin, Fällanden, Switzerland). Chemical shifts of 1H were referenced to 4,4-dimethyl-4-silapentane-1-sulfonic acid (0 ppm), and 13C chemical shifts were referenced indirectly using the gyromagnetic ratios of 13C and 1H (γ13C/γ1H = 0.25144952). All NMR data were processed using NMR Pipe [31 (link)] and were analyzed using CCPNMR [32 (link)].
Avance 800
Manufactured by Bruker
Sourced in Germany, United States
The Avance 800 is a high-performance nuclear magnetic resonance (NMR) spectrometer designed for advanced applications in chemistry, material science, and life sciences research. It offers a magnetic field strength of 800 MHz, providing high-resolution data for complex molecular structure determination and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Avance 500, by Bruker (5 mentions)
Avance 600, by Bruker (3 mentions)
Dmx 500, by Bruker (3 mentions)
Avanceiii 900, by Bruker (2 mentions)
Topspin, by Bruker (2 mentions)
5 mm triple resonance cryogenic probe, by Bruker (2 mentions)
Topspin 3, by Bruker (2 mentions)
Topspin 2, by Bruker (2 mentions)
Inova 600, by Agilent Technologies (2 mentions)
Cryogenic probe, by Bruker (1 mentions)
1h 13c 15n tci cryoprobe, by Bruker (1 mentions)
Drx 600 mhz spectrometer, by Bruker (1 mentions)
241 polarimeter, by PerkinElmer (1 mentions)
Microtof q esi mass spectrometer, by Bruker (1 mentions)
Avance 800 spectrometer, by Bruker (1 mentions)
Nmrpipe, by Bruker (1 mentions)
Sparky 3, by Bruker (1 mentions)
Unity inova 600 mhz, by Agilent Technologies (1 mentions)
Topspin 3.5pl2, by Bruker (1 mentions)
Drx 600, by Bruker (1 mentions)
40 protocols using avance 800
1
NMR Analysis of Fab/Fc and IgG Glycoproteins
2
NMR Analysis of FVIII-MCFD2 Interactions
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NMR measurements were performed using AVANCE 800 and AVANCE 500 spectrometers each equipped with a 5-mm triple-resonance cryogenic probe (Bruker BioSpin, Fallanden, Switzerland). The NMR data were processed and analyzed using TopSpin (Bruker BioSpin) and SPARKY software26 .
The chemical shifts of the FVIII fragment (Asn776–Asp838) were assigned by a standard protocol18 (link),25 (link). To obtain the dissociation constant based on NMR peak attenuation, 0.1 mM [15N]FVIII fragment was titrated with 0.02–0.3 mM of MCFD2/ERGIC-53CRD or MCFD2 only in 20 mM 2-(N-morpholino) ethanesulfonic acid (MES; pH 6.0) containing 10 mM CaCl2, 150 mM NaCl, and 10% (v/v) D2O. Regarding [15N]MCFD2, NMR experiments were performed as described previously18 (link); two molar excess amounts of peptides corresponding to Asp926–Lys953 of FV and Asn776–Asp838 of FVIII were individually added to 0.2 mM [15N]MCFD2. These experiments were performed in the presence or absence of five molar excess amounts of a tridecapeptide derived from Arg44–His56 of ERGIC-53 or that from Arg31–Arg44 of ERGL.
The chemical shifts of the FVIII fragment (Asn776–Asp838) were assigned by a standard protocol18 (link),25 (link). To obtain the dissociation constant based on NMR peak attenuation, 0.1 mM [15N]FVIII fragment was titrated with 0.02–0.3 mM of MCFD2/ERGIC-53CRD or MCFD2 only in 20 mM 2-(N-morpholino) ethanesulfonic acid (MES; pH 6.0) containing 10 mM CaCl2, 150 mM NaCl, and 10% (v/v) D2O. Regarding [15N]MCFD2, NMR experiments were performed as described previously18 (link); two molar excess amounts of peptides corresponding to Asp926–Lys953 of FV and Asn776–Asp838 of FVIII were individually added to 0.2 mM [15N]MCFD2. These experiments were performed in the presence or absence of five molar excess amounts of a tridecapeptide derived from Arg44–His56 of ERGIC-53 or that from Arg31–Arg44 of ERGL.
3
Resonance Assignments of Apo-HasAp Proteins
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Sequential backbone assignments of
wt apo-HasAp were obtained at 32 °C with the aid of two- and
three-dimensional NMR experiments [1H–15N HSQC, HNCA, HN(CO)CA, HNCACB, and CBCA(CO)NH] conducted in a Varian
Unity Inova 600 NMR spectrometer equipped with a triple-resonance
probe. Given that the 1H–15N HSQC spectra
of Y75A and H83A apo-HasAp are very similar to that of wt apo-HasAp,
sequential backbone assignments for the two mutants were obtained
at 32 °C with the aid of 1H–15N
HSQC, HNCA, and HN(CO)CA experiments conducted in a Bruker Avance
800 spectrometer equipped with a 5 mm TCI 1H/13C/15N cryoprobe. Protein samples for three-dimensional
NMR data acquisition were in phosphate buffer (20 mM, 95% H2O, 5% D2O, pH 7.0) at concentrations ranging between 2.5
and 5.0 mM. Two- and three-dimensional NMR spectra were processed
using NMRPipe41 (link) and analyzed with Sparky.42 1H chemical shifts were referenced
to the proton reference of DSS at 0 ppm, while 15N and 13C shifts were referenced indirectly using ratios of 0.101329118
and 0.251449530, respectively.43 (link) The effect
of temperature on the 1H–15N HSQC spectra
of wt and mutant proteins was determined in a 600 MHz Bruker Avance
III spectrometer equipped with an inverse H/C/N triple-resonance probe,
using samples with concentrations in the range of 1.0–1.7 mM.
wt apo-HasAp were obtained at 32 °C with the aid of two- and
three-dimensional NMR experiments [1H–15N HSQC, HNCA, HN(CO)CA, HNCACB, and CBCA(CO)NH] conducted in a Varian
Unity Inova 600 NMR spectrometer equipped with a triple-resonance
probe. Given that the 1H–15N HSQC spectra
of Y75A and H83A apo-HasAp are very similar to that of wt apo-HasAp,
sequential backbone assignments for the two mutants were obtained
at 32 °C with the aid of 1H–15N
HSQC, HNCA, and HN(CO)CA experiments conducted in a Bruker Avance
800 spectrometer equipped with a 5 mm TCI 1H/13C/15N cryoprobe. Protein samples for three-dimensional
NMR data acquisition were in phosphate buffer (20 mM, 95% H2O, 5% D2O, pH 7.0) at concentrations ranging between 2.5
and 5.0 mM. Two- and three-dimensional NMR spectra were processed
using NMRPipe41 (link) and analyzed with Sparky.42 1H chemical shifts were referenced
to the proton reference of DSS at 0 ppm, while 15N and 13C shifts were referenced indirectly using ratios of 0.101329118
and 0.251449530, respectively.43 (link) The effect
of temperature on the 1H–15N HSQC spectra
of wt and mutant proteins was determined in a 600 MHz Bruker Avance
III spectrometer equipped with an inverse H/C/N triple-resonance probe,
using samples with concentrations in the range of 1.0–1.7 mM.
4
NMR Spectroscopy of α-Synuclein Interactions
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Spectra from membrane-binding experiments were acquired using 0.1 mM 15N-enriched FL- and CT-α-syn dissolved in NMR buffer (20 mM HEPES, 100 mM KCl, pH7.0) plus 10% (v/v) D2O in the absence or presence of 12 mM liposomes. 1H-15N HSQC spectra from PDI-binding experiments were acquired using 0.3 mM 15N-enriched FL- and CT-α-syn dissolved in 20 mM HEPES, 100 mM NaCl, 5 mM tris (2-carboxyethyl) phosphine, pH 7.0, 10% (v/v) D2O in the absence, or presence of PDI at mole ratios from 10:1 to 1:1. Experiments were carried out at 15 °C on a Bruker Avance 800 or 850 MHz NMR spectrometer. Resonance assignments for α-syn are available from the BioMagResBank (entry number 16543). Data were analyzed with Sparky software. Intensity ratios were calculated by peak heights in the presence and absence of liposomes or PDI. CSPs of backbone amides were calculated according to Eq. (1 )95 (link), where ΔδH and ΔδN denote the chemical shift difference in the absence and presence of PDI in the 1H and 15N dimension, respectively.
KD was calculated using the fitting function shown in Eq. (2 )96 (link): where Δδobs is the observed FL- or CT-α-syn chemical shift minus the free FL- or CT-α-syn shifts, [P]t represents the concentration of FL- or CT-α-syn (300 μM), and [L]t represents PDI concentration, from 0 to 300 μM.
KD was calculated using the fitting function shown in Eq. (
5
NMR Characterization of His-IMP2KH34
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NMR spectroscopy used purified His-IMP2KH34. 2D 1H 15N HSQC experiments were acquired on a Bruker DRX600MHz spectrometer. 3D deuterium decoupled gradient sensitivity enhanced triple resonance experiments [HNCO, HN(CA)CO, HNCA, HN(CO)CA, HN(CA)CB, and HN(COCA)CB] experiments were acquired either on a Varian INNOVA 600 or a Bruker Avance™ 800 spectrometer with non-uniform sampling. NMR data were processed using NMRpipe/NMRDraw52 (link), analyzed using both CCPN Analysis53 (link) and NMRFAM-Sparky54 (link). Chemical shifts were indirectly referenced to sodium 2,2-dimethyl-2-silanepentane-5-sulfonate (DSS), using absolute frequency ratios for the 1H signal55 (link).
6
Multi-dimensional NMR Spectroscopy of Labeled Proteins
7
Gephyronic Acid Biotin Conjugation
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To a stirred solution of gephyronic acid methylester 2 (8.3 mg, 17.1 μmol) in CH2Cl2 (2 mL) were added D-(+)-biotin 3 (8.4 mg, 34.4 μmol), DCC (8.1 mg, 39.4 μmol) and DMAP (1.0 mg, 8.5 μmol) at room temperature. After stirring for 12 days, the reaction mixture was filtered through celite and the solvent was removed under vacuum. Purification by column chromatography on silica gel 60 (grain size 0.04–0.063 mm, Fluka) and ethyl acetate/MeOH (10:1) as eluent provided 4a (0.8 mg, 1.1 μmol, 7%) and 4b (6.0 mg, 8.5 μmol, 50%) as colorless amorphous solids. 1H and 13C NMR spectra were recorded on Bruker Avance 600 (600 MHz and 150 MHz, respectively) and Avance 800 (800 MHz and 200 MHz, respectively) spectrometers. 13C NMR multiplicities were determined with DEPT experiments. The regioisomeric (C-11)-biotinylated compound 4a and (C-3)-biotinylated compound 4b were characterized by COSY, ROESY, HSQC and HMBC experiments. FTIR spectra were recorded on a Bruker Vektor 22 spectrometer. Mass spectra were measured on a Bruker Daltonics microTOF-Q ESI mass spectrometer. Optical rotations were measured on a Perkin-Elmer Polarimeter 241. Gephyronic acid methylester 2 was prepared according to literature procedures [7 (link)]. D(+)-Biotin 3 was commercially available.
8
NMR Spectroscopy of Labeled PAC3 Homodimer
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13C- and 15N-labeled non-tagged PAC3 homodimer (0.3 mM) and 15N-labeled non-tagged PAC3 homodimer (0.1 mM), dissolved in PBS (pH 6.8) containing 10% D2O (v/v), 1 mM EDTA, and 0.01% NaN3, were used for spectral assignment and relaxation experiments. All NMR data were acquired at 303 K using DMX-500, AVANCE-500, and AVANCE-800 spectrometers equipped with a 5-mm triple-resonance cryogenic probe (Bruker, Billerica, MA, USA). The NMR data were processed using TOPSPIN (Bruker) and NMRPipe [32 (link)]. Conventional 3D NMR experiments [33 (link)] were carried out for chemical shift assignments of the heteronuclear single-quantum correlation (HSQC) peaks originating from the PAC3 homodimer. Spectral assignments were carried out using SPARKY [34 ] and CCPNMR [35 (link)] software. 15N relaxation parameters, T1, T2, and 15N-1H heteronuclear nuclear Overhauser effect (NOE) were obtained at 303 K using an AVANCE-800 spectrometer and analyzed using the Protein Dynamics software in the Dynamics Center (Bruker).
9
Structure-Activity of HIV-1 RT-DNA Aptamers
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To determine the structure-activity relationship of HIV-1 RT complexed with DNA aptamers, an NMR experiment was performed following Thammaporn et al. [24 (link)]. NMR is an effective method for analyzing HIV-1 RT binding to drugs. The results show the 1H-13C heteronuclear single-quantum coherence (HSQC) data of HIV-1 RT-NNRTIs. The 1H-13C HSQC spectra of WT or KY HIV-1 RTs labeled at methyl-13C-methionine on the p66 subunit were measured in the presence and absence of DNA aptamers. The HIV-1 RT and DNA aptamers were prepared in 10 mM Tris-d11 buffer (pD 7.6) containing 200 mM KCl, 1.5 mM sodium azide, and 4 mM MgCl2. Twenty-eight micromolar HIV-1 RT was complexed with 140 mM DNA aptamers (1:5 molar ratio). The reaction was measured by using an AVANCE800 (Bruker BioSpin; Karlsruhe, Germany) spectrometer supplied with a cryogenic probe. The spectra data were prepared and analyzed by the Topspin 3.2 (Bruker BioSpin; Karlsruhe, Germany) and SPARKY 3.115 programs (San Francisco, CA, USA).
10
NMR Experiments at Precise Temperatures
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All NMR experiments were performed at 15°C using Bruker Avance 800-MHz spectrometers equipped with triple-resonance, 5-mm triple axis–shielded gradient probes. For titration experiments we used the Varian Unity INOVA 600 MHz spectrometer equipped with a triple-resonance, 5-mm triple-axis shielded gradient probe at 25°C.
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