Hcn cold probe

Manufactured by Agilent Technologies

The HCN cold probe is a specialized laboratory equipment designed for nuclear magnetic resonance (NMR) spectroscopy. It is optimized for the detection and analysis of hydrogen (H), carbon (C), and nitrogen (N) nuclei in samples. The core function of the HCN cold probe is to enhance signal-to-noise ratio in NMR experiments, enabling improved sensitivity and resolution in the detection and characterization of molecules and compounds.

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Lab products found in correlation

Av 500 spectrometer, by Bruker (1 mentions) Nano itc system, by TA Instruments (1 mentions) Dimethyl sulfoxide d6, by Cambridge Isotopes (1 mentions) Inova 800 mhz spectrometer, by Agilent Technologies (1 mentions) Vnmrs 800 mhz spectrometer, by Agilent Technologies (1 mentions) 800 mhz spectrometer, by Agilent Technologies (1 mentions) Mestrenova 10, by Mestrelab Research (1 mentions) Dd2 700 mhz spectrometer, by Agilent Technologies (1 mentions) Acrodisc syringe filter, by Pall Corporation (1 mentions) Supor membrane, by Pall Corporation (1 mentions) Vnmrj 4, by Agilent Technologies (1 mentions)

5 protocols using hcn cold probe

Synthesis and Characterization of Compound 1

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All reagents were purchased from Merck and used as such. Compound 1 was synthesized according to a literature procedure [28 (link)] and purity was assessed via ¹H NMR (Figure S1). Established protocols were used to obtain ¹⁵N-labeled and unlabeled Saccharomyces cerevisae cytochrome c C102T [30 (link),31 (link),32 (link)]. A Bruker AV500 spectrometer recorded ¹H NMR spectra and δ values were expressed in ppm relative to D₂O (4.79 ppm at 25 °C). A 600 MHz Varian spectrometer equipped with a HCN cold probe acquired ¹H–¹⁵N HSQC spectra with spectral widths of 16 ppm (¹H) and 40 ppm (¹⁵N). ITC investigations were performed using a standard volume Nano ITC system equipped with a Hastelloy cell (TA Instruments).

Volpi S., Doolan A., Baldini L., Casnati A., Crowley P.B, & Sansone F. (2022). Complex Formation between Cytochrome c and a Tetra-alanino-calix[4]arene. International Journal of Molecular Sciences, 23(23), 15391.

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NMR Characterization of Lassomycin Structure

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NMR data were acquired and processed as previously described (Rea et al., 2010 ; Sit et al., 2011 (link)). A Varian Inova 800-MHz spectrometer with a triple-resonance HCN cold probe and pulsed field gradients (PFGs) was used to record spectra. [¹³C, ¹⁵N]lassomycin was dissolved in dimethyl sulfoxide-d₆ (Cambridge Isotope Laboratories, Andover, MA), and the sample was heated to 40 °C for data collection. Table S2 lists the experimental parameters used to acquire the NMR spectra for lassomycin. Tables S3–S4 list the proton, backbone nitrogen and carbon chemical shift assignments of the peptide. The ¹⁵N-HSQC (Fig. S1A) gave reasonably well-dispersed peaks, with 12 out of 16 unique backbone NH signals observed, indicating that lassomycin holds a defined structure in solution. The backbone NH signals for Arg3, Leu5, Arg14 and Ile16 could not be definitively assigned due to spectral overlap. Most of the proton chemical shift assignments were made based on data from the HCCH-TOCSY, ¹³C-NOESYHSQC and ¹⁵N-TOCSYHSQC experiments. Most of the carbon and nitrogen chemical shift assignments were made based on the backbone experiments HNCACB and CBCA(CO)NH (Sit et al., 2011 (link)).

Gavrish E., Sit C.S., Cao S., Kandror O., Spoering A., Peoples A., Ling L., Fetterman A., Hughes D., Bissell A., Torrey H., Akopian T., Mueller A., Epstein S., Goldberg A., Clardy J, & Lewis K. (2014). Lassomycin, a ribosomally synthesized cyclic peptide, kills Mycobacterium tuberculosis by targeting the ATP-dependent protease ClpC1P1P2. Chemistry & biology, 21(4), 509-518.

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Structural Determination of Wss1-VIM Peptide

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The Wss1 VIM (209–219) peptide was synthesized in Anaspec. RMN data were acquired at 278 K on an Agilent VNMRS 800 MHz spectrometer equipped with a triple-resonance HCN cold probe and pulsed field gradients. The 1H frequencies assignment was achieved with the combined use of 1H-1H- DQF-COSY, TOCSY (mixing time 80 ms) and NOESY (mixing time: 250 ms) spectra. CYANA 2.1 was used to calculate the structure of Wss1-VIM peptide (Guntert et al., 1997 (link)), using NOE restraints measured from the 1H-1H NOESY spectrum. From the observation of NOE cross peaks characteristic of alpha helical conformation daN(i,i+2) daN(i,i+3) and dNN(i,i+2) (Wuthrich, 1986 ) and the chemical shift analysis of the Ha chemical shifts of the peptide using the program TALOS (Shen et al., 2009 (link)) showing helical propensities for all residues, we applied PHI and PSI dihedral angles restraints corresponding to canonical helix values along the peptide backbone. The automatically assigned NOEs were calibrated within CYANA according to their intensities. After seven rounds of calculation (10,000 steps per round), 120 cross-peak NOE assignments were used in the final calculation. The 10 lowest energy conformations of the peptide have no constraint violations and show a backbone root-mean-square deviation of 0.2 ± 0.1 Å and a heavy atom root- mean-square deviation of 0.8 ± 0.2 Å.

Balakirev M.Y., Mullally J.E., Favier A., Assard N., Sulpice E., Lindsey D.F., Rulina A.V., Gidrol X, & Wilkinson K.D. (2015). Wss1 metalloprotease partners with Cdc48/Doa1 in processing genotoxic SUMO conjugates. eLife, 4, e06763.

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Optimized NMR Pulse Sequences for Hsp90

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NMR data were acquired at 298 K with Bruker 900 MHz spectrometers equipped with a TCI CryoProbe. A U-[85% ²H ¹⁵N,¹³C]-labeled human heat shock protein 90 (Hsp90) sample (25 kDa) in 20 mM phosphate buffer at pH 7.0, 100 mM KCl and 5mM BME was used for testing all of the pulse sequences. All comparisons were conducted using identical acquisition and processing parameters. The 3D TROSY-HNCA comparison was performed with the standard Bruker pulse sequence trhncagp2h3d2^{4 (link)} and the updated one (described below). The 3D TROSY-HNCACB comparison was conducted between the reference Bruker pulse sequence (trhncacbgp2h3d2) and the updated one (described below).
The first 2D H-C planes of 3D ghn_ca, ghn_caB, and ghnca_trosy_3DA were acquired with an Agilent/Varian 800 MHz spectrometer equipped with an HCN coldprobe at NMRFAM. The ghn_ca and ghnca_trosy_3DA are pulse sequences included in the Biopack software, and the ghn_caB is a modified version of the ghn_ca sequence (Fig. S3). As test sample, we used a triply labeled (²H,¹³C,¹⁵N, >95% deuterated) protein of molecular weight 37 kDa at a concentration of 0.2 mM in 10 mM Tris buffer at pH 6.5, 50 mM NaCl and 10% D₂O.

Xia Y., Rossi P., Tonelli M., Huang C., Kalodimos C.G, & Veglia G. (2017). Optimization of 1H decoupling eliminates sideband artifacts in 3D TROSY-Based Triple Resonance Experiments. Journal of biomolecular NMR, 69(1), 45-52.

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NMR Analysis of Enzymatic Deacetylation

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¹H-NMR was performed to monitor the activity of FjoAcXE and AxyAgu115A on Ac-XOS substrates. Reactions comprised 1% (w/v) acetylated (glucurono)-xylooligosaccharides in 50 mM HEPES buffer (pH 7.0) and 10 µg of each protein; the final reaction volume was 400 µL. The reaction continued for 20 h at 30 °C and gentle shaking. Reaction mixtures without enzyme were used as negative controls. Following the incubation, samples were filtered through Acrodisc^® syringe filters with 0.2-µm Supor^® membrane (Pall Corporation), and lyophilized. The samples were then dissolved in 300 µL D₂O and transferred into 3-mm NMR tubes (Norell) for analysis using an Agilent DD2 700 MHz spectrometer equipped with a triple resonance HCN cold probe with a scan number of 64, relaxation delay of 1 s and acquisition time of 4.5 s. The data were obtained using VnmrJ 4.0 (Agilent) and analyzed with MestReNova 10.0 (Mestrelab Research). The HDO peak at 4.790 was used as internal standard. The change in signal intensity in the regions between 5.4 and 4.4 ppm corresponding to acetylated Xylp residues in the anomeric region of the spectrum, and 2.30–2.05 ppm region corresponding to the acetyl group methyl protons were used to assign proton chemical shifts, as reported in Uhliariková et al. [24 (link)].

Razeq F.M., Jurak E., Stogios P.J., Yan R., Tenkanen M., Kabel M.A., Wang W, & Master E.R. (2018). A novel acetyl xylan esterase enabling complete deacetylation of substituted xylans. Biotechnology for Biofuels, 11, 74.

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