Esp 300 spectrometer

X-band and Q-band continuous wave (CW) spectroscopies were conducted using a Bruker ESP 300 spectrometer and a Bruker EMX spectrometer, respectively, both equipped with continuous helium flow Oxford cryostats. Photolysis was carried out for varying times using a 450 nm Thorlabs diode laser situated ≲1 cm from samples in the EPR cavity maintained at 12 K. Generally, the integrity of a SAM-bound cluster prior to photolysis was checked at 12 K, photolysis was performed, and then the temperature was raised to 40 K to characterize radical species. The modulation amplitude was normally set to 10 G, while the power was at 1 or 2 mW.

X-band continuous wave (CW) measurements were performed on a Bruker ESP 300 spectrometer with an ER 041 MR microwave bridge and an ER 4116DM resonator. All other EPR measurements were performed on a Bruker Elexsys E580 X-band spectrometer equipped with a SuperX-FT microwave bridge. For pulse EPR measurements at X-band, a Bruker ER 4118X-MS5 resonator was used in concert with an Oxford CF935 helium flow cryostat. Microwave pulses generated by the microwave bridge were amplified by a 1 kW traveling wave tube (TWT) amplifier (Applied Systems Engineering, model 117x). Pulse EPR spectra at Q-band frequencies were acquired using a home-built intermediate-frequency extension of the SuperX-FT X-band bridge that has a Millitech 5W pulse power amplifier. All experiments were conducted on a home-built TE₀₁₁ resonator utilizing the open resonator concept developed by Annino et al.⁶² and mechanical construction of the probehead similar to that presented by Reijerse et al.^{63 (link)} This setup allows t(π/2) = 12–16 ns at maximum input power with spectrometer dead time (including the resonator ring time) of 100–120 ns. Data acquisition and control of experimental parameters were performed by using Bruker XEPR software.

EPR spectra were obtained using a Bruker ESP 300 spectrometer (Bruker, Germany) equipped with a low-noise microwave amplifier (Miteq, Hauppauge, NY) and loop gap resonator (Medical Advances, Milwaukee, WI). The modulation amplitude was set at no greater than one-fourth of the line width. Spectra were collect at room temperature in the first-derivative mode.

Continuous-wave (CW) X-band (9.32 GHz) EPR spectra of 1 were collected on a modified Bruker ESP-300 spectrometer with 100 kHz field modulation (4 G modulation amplitude) at 20 K through the utilization of an Oxford Instruments liquid helium flow cryostat. Simulations of EPR spectra were performed using the MATLAB EasySpin (v4.5) toolbox (easyspin.org).^{17 (link)}

We used the pull down method to purify spin labeled trans-SNAREpin in between nanodiscs. Spin-labeled VAMP-2 was incorporated into the His-tag-free v-nanodisc and the unlabeled t-SNARE was incorporated into the 6× His-tagged t-nanodisc, respectively. The t-nanodiscs were attached to Ni-NTA sepharose beads and equal molar quantities of S-v-nanodiscs were then added and incubated for overnight at 4 °C to form spin labeled trans-SNAREpin. After washing out of unbound free S-v-nanodiscs spin labeled trans-SNAREpins were eluted from the beads by imidazole. To obtain spin labeled cis–SNARE complex in the nanodisc, the mixture of syntaxin-1a, 6× His-tagged SNAP-25, and spin-labeled VAMP-2 were incubated with molar ratio of 1:2:1. Unbound syntaxin-1a and spin-labeled VAMP-2 were washed out using the Ni-NTA column and the complex was then incorporated into the nanodisc. Each spin-labeled VAMP-2 was reconstituted into nanodiscs for the EPR measurement. EPR spectra were collected in the first-derivative mode with 1 mW microwave power using the Bruker ESP 300 spectrometer equipped with a loop-gap resonator. The modulation amplitude was set at no greater than one-fourth of the line width. All EPR measurements were performed at room temperature.