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900 mhz spectrometer

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The 900 MHz spectrometer is a high-performance laboratory instrument designed for nuclear magnetic resonance (NMR) analysis. It operates at a frequency of 900 MHz, providing a high-resolution platform for studying the structure and dynamics of chemical and biological samples.

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600 mhz spectrometer, by Agilent Technologies (1 mentions) Itc200, by Malvern Panalytical (1 mentions) Triple resonance cryoprobe, by Agilent Technologies (1 mentions)

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Spectrometers (10 citations)

10 protocols using 900 mhz spectrometer

1

Dynamics of Cyclophilin Proteins Studied by NMR

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15N TROSY CPMG-RD experiments were conducted with 0.5 mM 2H- and 15N-labeled CypA, CypB, CypC, or GeoCyp with 6 mM peptide substrate, 6 mM TA-peptide substrate, or 0.7 mM CsA. Data were collected on both a Varian 900 MHz spectrometer with a cryogenically cooled probe and a Varian 600 MHz spectrometer. Constant time relaxation periods of 60, 30, 40, and 70 ms were used for CypA, CypB, CypC, and GeoCyp, respectively. The R2 relaxation rate was calculated by31 (link) R2=−1Tlog(IτI0) where T is the constant time relaxation period, Iτ is the peak intensity at a given refocusing time τ, and I0 is the peak intensity in the absence of the constant time relaxation period. Exchange parameters were determined by least-squares fitting to the Carver–Richards equations, which describe generalized two-state exchange.21 R1 relaxation was measured using 0.5 mM N-labeled protein with mixing times of 10, 30, 50, 70, 90, and 110 ms. All data were collected at 10 °C.
Holliday M.J., Armstrong G.S, & Eisenmesser E.Z. (2015). Determination of the Full Catalytic Cycle among Multiple Cyclophilin Family Members and Limitations on the Application of CPMG-RD in Reversible Catalytic Systems. Biochemistry, 54(38), 5815-5827.
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2

NMR Characterization of Pin1 Protein

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The 15N,13C-labeled apo sample contained 2 mM Pin1 in 20 mM sodium phosphate, 50 mM sodium chloride, 5 mM dithiothreitol, 0.03% sodium azide, and 3% D2O at pH 6.5 through buffer exchange. NMR experiments were performed at 298 K on a triple-resonance Varian 900 MHz spectrometer equipped with a cryoprobe. The sequence specific backbone assignment was determined using 15N-HSQC, HNCACB, HNCA, CBCA(CO)NH, and CCONH spectra. For side-chain assignments, 13C-resolved aliphatic and aromatic CT-HSQC, HBHA(CO)NH, and HCCH-TOCSY spectra were used. All data was processed with NMRPipe (Delaglio et al. 1995 (link)) and analyzed using CCPNMR (Vranken et al. 2005 (link)). We observed no systematic deviation from the previously published chemical shifts of the isolated domains. For comparison of the shifts, we corrected the isolated-domain shifts by the average deviation from the full-length Pin1 shifts. After excluding those peaks with the strongest deviation, we recalibrated the isolated-domain shifts again for the final comparison.
Born A., Nichols P.J., Henen M.A., Chi C.N., Strotz D., Bayer P., Tate S.I., Peng J.W, & Vögeli B. (2018). Backbone and side-chain chemical shift assignments of full-length, apo, human Pin1, a phosphoprotein regulator with interdomain allostery. Biomolecular NMR assignments, 13(1), 85-89.
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3

Dynamic NMR Characterization of Cyclophilins

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15N TROSY CPMG-RD experiments were conducted with 1 mM deuterated, isotopically 15N-labeled CypA and GeoCyp on a Varian 900 MHz spectrometer with a cryogenically cooled probe. Data were collected at 0, 10, 20, and 30 °C for CypA, as indicated, using constant time relaxation periods of 50, 60, 80, and 90 ms. Data were collected at 0, 10, 20, and 30 °C for GeoCyp, using constant time relaxation periods of 60, 70, 90, and 100 ms. R1 relaxation was measured on 0.5 mM [15N]CypA or GeoCyp, using mixing times of 10, 30, 50, 70, 90, and 110 ms.
Holliday M.J., Camilloni C., Armstrong G.S., Isern N.G., Zhang F., Vendruscolo M, & Eisenmesser E.Z. (2015). Structure and Dynamics of GeoCyp: A Thermophilic Cyclophilin with a Novel Substrate Binding Mechanism That Functions Efficiently at Low Temperatures. Biochemistry, 54(20), 3207-3217.
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4

CypA Binding Affinity Measurement via NMR and ITC

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Binding affinity was measured via collection of 15N-HSQC spectra of 15N-CypA upon serial addition of unlabeled FGP, as described previously (Holliday et al., 2015b (link)). Spectra were collected on a Varian 900 MHz spectrometer upon addition of 0, 0.1, 0.2, 0.5, 1, and 2 mM unlabeled peptide to 0.5 mM 15N-CypA. For all non-overlapping residues, data were first fit individually to the steady-state equilibrium binding equation; data were then fit simultaneously for affected residues exhibiting fast exchange (identified by an individual fit with r2 > 0.99, between 46 and 50 residues per titration experiment). 15N and 1H chemical shifts were treated independently in this analysis. Binding affinity was measured for FGP, HA, and HAP2A via ITC. HA and HAP2A ITC data were collected using 0.5 mM protein and titrating in up to a 1:2 molar ratio of each peptide on a MicroCal iTC200 and processed using MicroCal ITC-ORIGIN. FGP ITC data were collected with 0.5 mM protein and titration up to a 1:5 molar ratio on a TA Nano ITC and processed with NanoAnalyze. All data were collected at 10°C.
Holliday M.J., Camilloni C., Armstrong G.S., Vendruscolo M, & Eisenmesser E.Z. (2017). Networks of Dynamic Allostery Regulate Enzyme Function. Structure (London, England : 1993), 25(2), 276-286.
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5

NMR Spectroscopy of Protein Complexes

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All NMR spectra were recorded at 25 °C on a triple-resonance Varian 900 MHz spectrometer equipped with a cryo-probe. The linear sampling spectra of OBP22 and Pin1 were acquired with 1024 complex points in the direct 1H dimension, and 56 and 72 complex points in the indirect 1H and 13C dimensions, respectively. Non-uniform sampling schemes were generated by the NUS@HMS generator software [11 (link)], with 1024 complex data points in the direct 1H dimension, and 50% sampling of the original 89 and 90 complex points in the 1H and 13C dimension, respectively, resulting in the same overall measurement times as those for linear sampling. The spectral widths for both the linear and NUS sampling spectra were 14,044.9 Hz (direct 1H), 10,793.3 Hz (indirect 1H), and 6785.4 (13C) with an interscan delay of 1.6 s, and eight scans. Linear prediction was performed on noted linearly sampled data with the order set to 28 in the indirect 1H dimension. The NUS-acquired data were reconstructed using the hmsIST software [11 (link)]. Zero filling was achieved by rounding the size to a power of two (auto) in both indirect dimensions. Both linear and NUS sampling experiments took 60 h for measurement.
Born A., Henen M.A., Nichols P., Wang J., Jones D.N, & Vögeli B. (2018). Efficient Stereospecific Hβ2/3 NMR Assignment Strategy for Mid-Size Proteins. Magnetochemistry (Basel, Switzerland), 4(2), 25.
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6

Protein-Ligand Binding Affinity Determination

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Binding affinity was measured by NMR titration. 15N HSQC spectra were collected on 500 μM 15N-labeled protein in the presence of 0, 0.1, 0.2, 0.5, 1, and 2 mM peptide substrate on a Varian 900 MHz spectrometer. For peaks with significant chemical shift changes upon titration, chemical shift changes were least-squares fit individually to the steady state equilibrium binding equation below.
F([L])=Fmax[P]+[L]+KD-([P]+[L]+KD)2-4[P][L]2[P] where F([L]) is the ligand-dependent chemical shift change, Fmax is the chemical shift change upon full saturation, [P] is the total protein concentration, [L] is the total ligand concentration, and KD is the dissociation constant. All chemical shifts that could be fit well individually (r2 > 0.99; indicating they are in the fast exchange regime needed to accurately calculate binding affinity) were then fit simultaneously, yielding a single dissociation constant determined for each protein.
Holliday M.J., Camilloni C., Armstrong G.S., Isern N.G., Zhang F., Vendruscolo M, & Eisenmesser E.Z. (2015). Structure and Dynamics of GeoCyp: A Thermophilic Cyclophilin with a Novel Substrate Binding Mechanism That Functions Efficiently at Low Temperatures. Biochemistry, 54(20), 3207-3217.
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7

NMR Spectroscopy of Protein Complexes

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All NMR spectra were recorded at 25 °C on a triple-resonance Varian 900 MHz spectrometer equipped with a cryo-probe. The linear sampling spectra of OBP22 and Pin1 were acquired with 1024 complex points in the direct 1H dimension, and 56 and 72 complex points in the indirect 1H and 13C dimensions, respectively. Non-uniform sampling schemes were generated by the NUS@HMS generator software [11 (link)], with 1024 complex data points in the direct 1H dimension, and 50% sampling of the original 89 and 90 complex points in the 1H and 13C dimension, respectively, resulting in the same overall measurement times as those for linear sampling. The spectral widths for both the linear and NUS sampling spectra were 14,044.9 Hz (direct 1H), 10,793.3 Hz (indirect 1H), and 6785.4 (13C) with an interscan delay of 1.6 s, and eight scans. Linear prediction was performed on noted linearly sampled data with the order set to 28 in the indirect 1H dimension. The NUS-acquired data were reconstructed using the hmsIST software [11 (link)]. Zero filling was achieved by rounding the size to a power of two (auto) in both indirect dimensions. Both linear and NUS sampling experiments took 60 h for measurement.
Born A., Henen M.A., Nichols P., Wang J., Jones D.N, & Vögeli B. (2018). Efficient Stereospecific Hβ2/3 NMR Assignment Strategy for Mid-Size Proteins. Magnetochemistry (Basel, Switzerland), 4(2), 25.
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8

NMR Titration of Cyclophilins

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15N HSQC spectra were collected on 0.5 mM 15N-labeled CypA, CypB, CypC, or GeoCyp in the presence of 0, 0.1, 0.2, 0.5, 1, or 2 mM unlabeled peptide substrate. Titration data were collected at 10 °C on a Varian 900 MHz spectrometer with a cryogenically cooled probe and fit as previously described.24 (link)
Holliday M.J., Armstrong G.S, & Eisenmesser E.Z. (2015). Determination of the Full Catalytic Cycle among Multiple Cyclophilin Family Members and Limitations on the Application of CPMG-RD in Reversible Catalytic Systems. Biochemistry, 54(38), 5815-5827.
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9

NMR Analysis of TLR5-HMGB1 Interactions

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drTLR5/HMGB1 interactions were determined by solution NMR spectroscopy on the CBP- and 6X His tags containing full-length and tailless 15N uniformly labeled HMGB1 proteins ranging from 80–100 µM, 10% (v/v) 2H20 in 10 mM Tris HCl (pH 8.0) and 1 mM deuterated DTT. Titrations of 1:1 and 1:2 (mol/mol) drTLR5/HMGB1 (CBP- and 6X His tag containing) were used All spectra were collected either in a Varian 800 MHz or 900 MHz spectrometer equipped with triple resonance cryoprobe at 25°C housed at Univ. Colorado Boulder and Denver campuses.
Das N., Dewan V., Grace P.M., Gunn R.J., Tamura R., Tzarum N., Watkins L.R., Wilson I.A, & Yin H. (2016). HMGB1 activates proinflammatory signaling via TLR5 leading to allodynia. Cell reports, 17(4), 1128-1140.
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10

NMR Analysis of Protein Complexes

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NMR samples were prepared in the above defined NMR buffer, and all 1D and 2D spectra presented here were collected on a Varian 900 MHz spectrometer (Agilent Technologies, Pasadena, CA, USA) equipped with a cryo-probe at 25 °C, unless otherwise described. As IL-1R8-TIR was stable for only several hours, 10% glycerol was added to increase the lifespan of each sample to several weeks as monitored through 15N-HSQC spectra. For 1D spectra that followed NADase activities, first the indicated concentrations of NAD+ and products were incubated, and the specified TIR-containing proteins were added prior to collection, with a total of 16 scans averaged over each minute. For relaxation data, standard Varian BioPack R1 and R2 relaxation experiments were collected on a Varian 900 MHz spectrometer. Assignment experiments for 2H,13C,15N-labeled tirE-CC were collected on a Bruker 800 equipped with a cryo-probe at 25 °C, which included an HNCACB, HNcoCACB, HNCO, and HNCACO. Assignment experiments for 2H,13C,15N-labeled IL1-R8-TIR were collected on a Varian 600 equipped with a cryo-probe at 30 °C, which included an HNCACB, HNcoCACB, HNCO, and HNCACO. Assignments for tirE-CC and IL1-R8-TIR have been deposited in the Biological Magnetic Resonance Bank with the accession codes 51474 and 51473, respectively.
Lee E., Redzic J.S., Nemkov T., Saviola A.J., Dzieciatkowska M., Hansen K.C., D’Alessandro A., Dinarello C, & Eisenmesser E.Z. (2022). Human and Bacterial Toll-Interleukin Receptor Domains Exhibit Distinct Dynamic Features and Functions. Molecules, 27(14), 4494.
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