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8 protocols using er4122shqe

1

Copper-Protein EPR Spectroscopy Protocol

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All samples were made to pH 6.0 in 50 mM 2-(N-morpholino)ethanesulfonic acid (Mes) buffer (Sigma), using potassium as a counterion. The protein was added to a concentration of 100 μM, and CuCl2 was used at 100 μM. The samples contained 30% glycerol as a cryoprotectant. X-band (9.38 GHz) continuous-wave EPR spectra were recorded on a Bruker EleXsys E580 spectrometer equipped with a super-high Q resonator (ER4122SHQE). Cryogenic temperatures were achieved with a liquid nitrogen finger Dewar and gas flow controller. The spectrometer settings were as follows: temperature = 121 K, conversion time = 41 ms, modulation amplitude = 0.5 mT, modulation frequency = 100 kHz, bridge power = 5 mW, attenuation = 23 dB.
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2

Continuous Wave EPR Experiments

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Continuous wave (cw) X−band (9–10 GHz) EPR experiments were carried out with Bruker ELEXSYS E580 and ELEXSYS E500 II EPR spectrometers (Bruker Biospin, Rheinstetten, Germany), equipped with an Bruker ER4102ST resonator, ER4122SHQE resonator, or Flexline dielectric ring resonator (Bruker ER 4118X-MD5-W1). Helium gas-flow cryostats (Oxford Instruments and ICE Oxford, UK) and an ITC (Oxford Instruments, UK) were used for cryogenic temperatures. Light excitation was done directly in the resonator with 532 nm Laser light (Nd:YAG Laser, INDI, Newport) or with a white light LED (Thorlabs).
High frequency (HF) EPR measurements were performed on a home-built D-band (130 GHz) spectrometer equipped with a single mode TE011 cylindrical cavity.51 –52 (link) D-band EPR spectra were recorded in pulse mode in order to remove the microwave phase distortion due to fast-passage effects at low temperatures. Light excitation was done directly in the cavity of the spectrometer with 532 nm Laser light through an optical fiber (Nd:YAG Laser, INDI, Newport). Data processing was done using Xepr (Bruker BioSpin, Rheinstetten) and MatlabTM 7.11.2 (MathWorks, Natick) environment. Simulations of the EPR spectra were performed using the EasySpin software package.53 (link)
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3

CW-ESR Spectroscopy of Glycerol-Containing Samples

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For the CW-ESR measurement, ∼200 μL of the sample, which contained 20% glycerol, was transferred into a 4-mm EPR quartz tube. The CW-ESR spectra were recorded in a Bruker ELEXSYS 580, equipped with an X-band microwave bridge and ER 4122 SHQE resonator, and an ER 4131 VT unit for temperature control. The spectra were recorded at 200 K with a microwave frequency of ∼9.43 GHz, microwave power of 1.5 mW, modulation frequency of 100 kHz, modulation amplitude of 1 G, and scan width of 200 G and 1,024 data points.
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4

Cw-ESR Spectroscopy of Samples

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The cw-ESR measurements were performed at room temperature (RT), 296 K or low temperature, 163 K, on a Bruker ELEXIS E500 (Bruker, Billerica, USA) spectrometer equipped with a Bruker ER 4122SHQE resonator and Bruker VT-31 temperature controller. For the RT measurements the microwave power used was 1.26 mW and the modulation amplitude was 2.2 G. Low-temperature spectra were recorded at 0.05 mW microwave power and modulation amplitude of 2 G.
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5

CW EPR Spectroscopy of Paramagnetic Samples

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X-band (9.37 GHz) continuous-wave (CW) EPR spectra were recorded on a Bruker (Billerica, MA) EleXsys E500 spectrometer equipped with a super-high Q resonator (ER4122SHQE). Cryogenic temperatures were achieved and controlled using an ESR900 liquid helium cryostat in conjunction with a temperature controller (Oxford Instruments ITC503) and a gas flow controller. CW EPR data were collected under slow-passage conditions. The spectrometer settings were as follows: conversion time = 40 ms, modulation amplitude = 0.5 mT, and modulation frequency = 100 kHz; other settings are given in the corresponding figure captions. Spin quantification was determined by comparison of the double integral intensity of the EPR spectra to that of a standard solution of 100 μM CuSO4 with 200 μM HC1, 200 mM NaClO4 and 20% ethylene glycol. Simulations of the CW spectra and the following pulsed EPR spectra were performed using the EasySpin 5.1.10 toolbox39 (link),40 (link) within the Matlab 2014a software suite (The Mathworks Inc., Natick, MA). Euler angles follow the zyz convention.
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6

EPR Spectroscopy of HGA Oxidation

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CW (Continuous wave) X-band (9 GHz) EPR spectra of 45 μL HGA 30 mM and 5 μL NaOH 1 N were recorded at room temperature. The reaction was monitored at different reaction times after the addition of reagents. The reaction was also tested at acidic pH after the addition of HCl 1 N and in buffer solution at pH = 8. EPR measurements were performed with a Bruker E580 Elexsys Series using the Bruker ER4122SHQE cavity filling in a 1 mm ID quartz capillary tube and then it was placed inside standard suprasil EPR tubes. EPR spectra simulations were performed with the Easyspin software package30 (link), using the "garlic function".
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7

Intracellular Spin-Label Reduction Kinetics

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All cw-EPR experiments were acquired using a Bruker ELEXYS E580 spectrometer equipped with a HIGH Q ER4122SHQE operating at X-band using EPR quartz tube with 1.6 mm O.D and 1 mm I.D. The spectroscopic setting for the in vitro measurements was kept as following: ν = 9.874 GHz; center field = 3500 G; sweep width = 150 G; microwave power = 126 mW; modulation frequency = 100 kHz; modulation amplitude = 1 G; conversion time = 25 ms; sweep time = 25.6 s; scans = 25).
All in cell samples were recorded in a time resolved set-up with the same conditions except that a time axis was defined to follow the entire reduction of the spin-label inside the intracellular environments. Each experiment was acquired in 13 min.
The start of the measurements was in average 12–14 min after the last thermal treatment, and the zero time was set as the starting time of the delivery protocol.
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8

CW-EPR Spectroscopy of Powder Samples

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Room temperature X-band (9.8 GHz) CW-EPR spectra were collected on a Bruker EMX equipped with a super-high Q resonator (ER4122 SHQE). Low temperature Q-band (33.8 GHz) CW-EPR spectra were collected on a Bruker ELEXSYS E580 using an EN 5107D2 Bruker resonator housed in an Oxford CF935 cryostat. All samples were finely grinded in order to obtain a powder averaged spectrum. Spectra simulations were performed using EasySpin 5.2.28 toolbox 37 within the Matlab 2019b software suite (The Mathworks Inc., Natick, MA).
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