Average 15N relaxation times were determined from 1D 15N-edited T1 and T2 (CPMG) experiments on U-[13C, 15N]-labeled ZF4-5 recorded at a Varian Inova 600 MHz at 298 K. Longitudinal T1 relaxation delays were 50, 100, 200, 300, 400, 600, 800, 1000, 1500, and 2000 ms; transverse T2 relaxation CPMG delays were 10, 20, 30, 50, 70, 100, 130, 170, 210, and 250 ms; both experiments had 1.5 s recycle delays. T1 and T2 relaxation times were obtained by integration from 8.5 to 10.5 ppm, and τc was approximated following the literature equation.7 (link) The isotropic overall rotational correlation time of U-[13C, 15N]-labeled ZF4-5 was 7.3 ns based on the backbone 15N T1 and T2 relaxation time measurements. From the linear fit of protein molecular weight (M.W.) versus correlation time for a series of standard proteins,8 (link) ZF4-5 was estimated to have a M.W. of 9.6 kDa (Supporting Information Fig. S1 ). This result indicated that INSM1 ZF4-5 under the NMR study conditions existed predominantly as a monomer (unlabeled ZF4-5 M.W. of 9.2 kDa).
Inova 600 mhz
Manufactured by Agilent Technologies
Sourced in United States
The Inova 600 MHz is a high-performance nuclear magnetic resonance (NMR) spectrometer designed for analytical laboratory applications. It operates at a magnetic field strength of 600 MHz, providing enhanced resolution and sensitivity for the analysis of chemical structures and compositions.
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Lab products found in correlation
C18 column, by Phenomenex (2 mentions)
Cryogenic probe, by Agilent Technologies (1 mentions)
Ltq orbitrap xl, by Thermo Fisher Scientific (1 mentions)
Inova 400 mhz, by Agilent Technologies (1 mentions)
Synergy h4 hybrid multi mode microplate reader, by Synergy Software (1 mentions)
Hp 1100 lc system, by Phenomenex (1 mentions)
6520 q tof mass spectrometer, by Agilent Technologies (1 mentions)
Inova 700 mhz spectrometer, by Agilent Technologies (1 mentions)
Zorbax rx c8, by Agilent Technologies (1 mentions)
Spectramax m5 spectrophotometer, by Molecular Devices (1 mentions)
Nmr spectrometer, by Agilent Technologies (1 mentions)
Mercury 400 mhz, by Agilent Technologies (1 mentions)
Inova 300 mhz, by Agilent Technologies (1 mentions)
Zq 4000 mass spectrometer, by Waters Corporation (1 mentions)
14 protocols using inova 600 mhz
1
Measuring Protein Dynamics Using NMR
2
NMR Characterization of RHE_CH02687
3
Monitoring IFNA2 Amide Shifts by 2D HSQC
4
NMR Spectroscopy for Product Characterization
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NMR spectra were recorded at 25 °C using Inova 400 MHz and Inova 600 MHz, equipped with a cryogenic probe optimized for 1H detection, spectrometers (Varian Inc., Palo Alto, CA, USA). Chemical shifts were referenced to internal Tetramethylsilane (TMS). Reactions were monitored by mono-dimensional (1D) 1H NMR spectra. Products were characterized by 1D and bi-dimensional (2D) homo- and hetero-nuclear spectra. Homo-nulclear 1H-1H scalar correlations were determined by TOCSY (Total Correlation Spectroscopy) experiments with mixing time of 70 ms. One-bond heteronuclear 1H-13C correlations were determined by HSQC (Heteronuclear Single Quantum Coherence) experiments. Two and three-bond heteronuclear 1H-13C correlations were determined by HMBC (Heteronuclear Multiple Bond Correlation) experiments with gradients of refs 2 (link) and 3 (link) J from 5 to 15 Hz. Low- and high-resolution ESI-MS spectra were performed on a LTQ Orbitrap XL (ThermoScientific) mass spectrometer.
5
NMR, Mass Spectrometry, and HPLC Analysis
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Unless otherwise indicated, all starting materials, chemicals, and anhydrous solvents were purchased from Sigma Aldrich (NSW, Australia) and were used without further purification. 1H and 13C-NMR spectra were recorded on a Varian 500 MHz or a Varian Inova 600 MHz instruments in the indicated solvents. Chemical shifts are reported in ppm (δ). Signals are reported as s (singlet), br s (broad singlet), d (doublet), dd (doublet of doublets), t (triplet), or m (multiplet). High resolution mass spectra were collected using an LTQ Orbitrap XL ETD using flow injection, with a flow rate of 5 μL/min. Where indicated compounds were analyzed and purified by reverse phase HPLC, using an HP 1100 LC system equipped with a Phenomenex C-18 column (250 × 4.6 mm) for analytical traces and a Gilson GX-Prep HPLC system equipped with a Phenomenex C18 column (250 × 21.2 mm). H2O and MeCN solutions were used as aqueous and organic buffers. All absorbance and fluorescence spectra were collected on a Synergy H4 Hybrid Multimode Microplate Reader using black-walled clear bottom 96-well plates. Data was processed using Microsoft Excel 2016 and all graphs were generated using GraphPad Prism 7 software. A mercury lamp (365 nm) was used as the UV light source in photoswitching experiments.
6
NMR Spectroscopy of TTD Binding
7
Determining Rotational Correlation Time of CGL2373
8
NMR spectroscopy of matrilin-4 VWA2 domains
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A 15N 13C labeled 100 μM mouse matrilin-4 VWA2 domain was prepared in in 20 mM Bis–Tris, 150 mM KCl, 1 mM NEM, pH 7 and a 15N 13C labeled 300 μM zebrafish matrilin-4 VWA2 domain was prepared in 20 mM Bis–Tris, 150 mM KCl, pH 7, respectively. NMR experiments with the murine domain were performed at 25 °C on a Varian Inova 600 MHz (1H) instrument equipped with a z-gradient triple resonance room temperature probe. 2D 1H–15N and 1H–13C HSQC spectra incorporating gradient enhanced coherence selection and water flip back were recorded with acquisition times of 63 ms (t1, 15N) and 64 ms (t2, NH) and 16 ms (t1, 13C) and 51 ms (t2, H), respectively, using standard pulse sequences as implemented in BioPack (Varian, Palo Alto, US). NMR-experiments with the zebrafish VWA domain were performed at the Medical Research Council in Mill Hill (London, UK) at a Bruker Avance 700 MHz (1H) instrument equipped with a z-gradient cryoprobe. 2D 1H–15N HSQS spectra were recorded with acquisition times 104 ms (1H) and 47 ms (15N).
Data were processed using water suppression and mild resolution enhancement using NMRPipe [29] (link) and were analyzed with NMRView [30] (link).
Data were processed using water suppression and mild resolution enhancement using NMRPipe [29] (link) and were analyzed with NMRView [30] (link).
9
Synthesis and Characterization of Compound 4
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Unless otherwise indicated, all
starting materials, chemicals, and anhydrous solvents were purchased
from Sigma-Aldrich (Australia) and were used without further purification.
UNIBOND C-18 reverse-phase silica gel was purchased from Analtech
Inc. Compound4 (Scheme 1 ) was prepared as previously described.39 (link)1H and 13C NMR spectra
were recorded on a Varian 500 MHz and Varian Inova 600 MHz instruments
in the indicated solvents. Chemical shifts are reported in ppm (δ).
Signals are reported as s (singlet), brs (broad singlet), d (doublet),
dd (doublet of doublets), t (triplet), or m (multiplet). High-resolution
mass spectra were collected using an LTQ Orbitrap XL ETD with flow
injection, with a flow rate of 5 μL min–1.
Where indicated, compounds were analyzed and purified by reverse-phase
HPLC, using an HP 1100 LC system equipped with a Phenomenex C-18 column
(250 × 4.6 mm2) for analytical traces and a Gilson
GX-Prep HPLC system equipped with a Phenomenex C18 column (250 ×
21.2 mm2). H2O and acetonitrile solutions were
used as aqueous and organic buffers. All graphs were generated using
GraphPad Prism 7 software and IgorPro 6. A mercury lamp (365 nm) and
a halogen lamp (>450 nm) were used as the UV and visible light
sources in photoswitching experiments.
starting materials, chemicals, and anhydrous solvents were purchased
from Sigma-Aldrich (Australia) and were used without further purification.
UNIBOND C-18 reverse-phase silica gel was purchased from Analtech
Inc. Compound
were recorded on a Varian 500 MHz and Varian Inova 600 MHz instruments
in the indicated solvents. Chemical shifts are reported in ppm (δ).
Signals are reported as s (singlet), brs (broad singlet), d (doublet),
dd (doublet of doublets), t (triplet), or m (multiplet). High-resolution
mass spectra were collected using an LTQ Orbitrap XL ETD with flow
injection, with a flow rate of 5 μL min–1.
Where indicated, compounds were analyzed and purified by reverse-phase
HPLC, using an HP 1100 LC system equipped with a Phenomenex C-18 column
(250 × 4.6 mm2) for analytical traces and a Gilson
GX-Prep HPLC system equipped with a Phenomenex C18 column (250 ×
21.2 mm2). H2O and acetonitrile solutions were
used as aqueous and organic buffers. All graphs were generated using
GraphPad Prism 7 software and IgorPro 6. A mercury lamp (365 nm) and
a halogen lamp (>450 nm) were used as the UV and visible light
sources in photoswitching experiments.
10
Spectroscopic Characterization of Organic Compounds
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Optical rotation measurements were obtained on a Perkin-Elmer 241 Polarimeter calibrated using a Rudolph Quartz Control Plate Calibration Standard at sodium D line (at +11.502°). Ultraviolet spectra were obtained on a UV-visible Molecular Devices SpectraMax M5 spectrophotometer using 1-ml cuvettes with 1.0 cm path lengths at room temperature in solvent methanol (MeOH). Spectrophotometric assays were performed on Molecular Devices SpectraMax M5 384 variable wavelength spectrometer. All NMR spectra were acquired on a Varian INOVA 600 MHz and a Varian INOVA 700 MHz spectrometer at the NMR Facility, Department of Chemistry, University of Michigan. HRESIMS spectra were measured at the University of Michigan core facility in the Department of Chemistry using an Agilent 6520 Q-TOF mass spectrometer equipped with an Agilent 1290 HPLC system. RP-HPLC was performed using Econosil C18 10 μm 22 × 250-mm column and Agilent ZORBAX RX-C8 5 μm 9.4 × 250-mm column and a solvent system of MeCN and H2O. The LC-MS analysis of HPLC fractions was performed on a Shimadzu 2010 EV APCI spectrometer.
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