The final concentration of the spin-labeled
T4L was 0.3–0.5 mM in buffer A containing 30% (v/v) glycerol
(which is equivalent to the 10 mol % glycerol/water condition, approximately).
Oxygen was removed from the sample by using a freeze–thaw method.
Approximately, 40 μL of sample volume was loaded into two capillaries
(Kimble, 34507-99) prior to being placed in 4 mm quartz ESR tube.
All ESR measurements were carried out on a Bruker ELEXSYS E580 spectrometer
equipped with X-band microwave bridge (microwave frequency, 9.45 GHz),
an ER 4122 SHQE cavity, and an ER 4131 VT unit for temperature control.
For cw-ESR measurements, spectra were acquired with microwave power
of 1.5 mW, 100 kHz modulation frequency, 1 G modulation amplitude,
and 200 G sweep width. For ST-ESR measurements, spectra were acquired
according to the reported procedure,23 (link) with
90 mW incident microwave power, 50 kHz modulation frequency, 5 G modulation
amplitude, and 150 G sweep width. Modulation phase was set at 90°
with phase-sensitive detection at second harmonic. Null phase method
was used to minimize the signal at unsaturating microwave power.29 (link) For the measurements at increasing temperatures,
the ESR probe-head was precooled to 180 K prior to the transfer of
ESR sample tube into the cavity. Spectra were recorded stepwise from
180 to 240 K with an equilibrium time >10 min for each temperature.
T4L was 0.3–0.5 mM in buffer A containing 30% (v/v) glycerol
(which is equivalent to the 10 mol % glycerol/water condition, approximately).
Oxygen was removed from the sample by using a freeze–thaw method.
Approximately, 40 μL of sample volume was loaded into two capillaries
(Kimble, 34507-99) prior to being placed in 4 mm quartz ESR tube.
All ESR measurements were carried out on a Bruker ELEXSYS E580 spectrometer
equipped with X-band microwave bridge (microwave frequency, 9.45 GHz),
an ER 4122 SHQE cavity, and an ER 4131 VT unit for temperature control.
For cw-ESR measurements, spectra were acquired with microwave power
of 1.5 mW, 100 kHz modulation frequency, 1 G modulation amplitude,
and 200 G sweep width. For ST-ESR measurements, spectra were acquired
according to the reported procedure,23 (link) with
90 mW incident microwave power, 50 kHz modulation frequency, 5 G modulation
amplitude, and 150 G sweep width. Modulation phase was set at 90°
with phase-sensitive detection at second harmonic. Null phase method
was used to minimize the signal at unsaturating microwave power.29 (link) For the measurements at increasing temperatures,
the ESR probe-head was precooled to 180 K prior to the transfer of
ESR sample tube into the cavity. Spectra were recorded stepwise from
180 to 240 K with an equilibrium time >10 min for each temperature.