Spin-label CW microwave power saturation and rapid passage with triangular non-adiabatic rapid sweep (NARS) and adiabatic rapid passage (ARP) EPR spectroscopy

Abstract

Non-adiabatic rapid passage (NARS) electron paramagnetic resonance (EPR) spectroscopy was introduced by Kittell et al. (2011) as a general purpose technique to collect the pure absorption response. The technique has been used to improve sensitivity relative to sinusoidal magnetic field modulation, increase the range of inter-spin distances that can be measured under near physiological conditions (Kittell et al., 2012), and enhance spectral resolution in copper (II) spectra (Hyde et al., 2013). In the present work, the method is extended to CW microwave power saturation of spin-labeled T4 Lysozyme (T4L). As in the cited papers, rapid triangular sweep of the polarizing magnetic field was superimposed on slow sweep across the spectrum. Adiabatic rapid passage (ARP) effects were encountered in samples undergoing very slow rotational diffusion as the triangular magnetic field sweep rate was increased. The paper reports results of variation of experimental parameters at the interface of adiabatic and non-adiabatic rapid sweep conditions. Comparison of the forward (up) and reverse (down) triangular sweeps is shown to be a good indicator of the presence of rapid passage effects. Spectral turning points can be distinguished from spectral regions between turning points in two ways: differential microwave power saturation and differential passage effects. Oxygen accessibility data are shown under NARS conditions that appear similar to conventional field modulation data. However, the sensitivity is much higher, permitting, in principle, experiments at substantially lower protein concentrations. Spectral displays were obtained that appear sensitive to rotational diffusion in the range of rotational correlation times of 10(-3) to 10(-7) s in a manner that is analogous to saturation transfer spectroscopy.

Keywords: Adiabatic rapid passage; EPR; NARS; Nitroxide; Oxygen accessibility; Paramagnetic ion accessibility; Power saturation; Saturation transfer; Spin-label.

Figure 1

Rapid passage effects can be investigated using the NARS technique by comparing the ‘up-field sweep’ of the triangle to the ‘down-field sweep’.

Figure 2

Power saturation curves of T4L as a function of temperature acquired from the peak-to-peak center line height in spectra recorded with magnetic field modulation (A) and from the center peak height acquired with NARS detection (B); 260 kG/s, up-field sweep.

Figure 3

NARS (up-sweep, 260 kG/s) and CW spectra collected under nitrogen or in the presence of air at 30°C.

Figure 4

NARS (black, up-field sweep, 260 kG/s) and CW (red) EPR spectra of T4- Lysozyme under non-saturating (-32 dB, 53 μW) and moderate saturating (-17 dB, 1.9 mW) conditions. Rotational correlation times of 1.7 μs (−30°C), 0.26 μs (−10°C), and 0.077 μs (10°C) were estimated using the Debye expression. Spectra were normalized to the lowest microwave power, as in Fig. 2.

Figure 5

Resolution enhancement by differential saturation at −10°C (130 kG/s). A) Raw NARS spectra collected under non-saturating (-32 dB), moderate saturating (-20 dB), and saturating conditions (-8 dB). B) NARS spectra collected at two microwave powers, adjusting the amplitude of the lower power to account for the expected amplitude difference due to the change in microwave power (dashed). C) The difference of the black spectrum from the dashed green spectrum in Panel B in shown in red. MDIFF was applied to the red spectrum to enhance turning points in the lower trace.

Figure 6

(A) NARS spectra of T4L acquired on the up- and down-sweeps of the triangle at −30°C with a 260 kG/s sweep rate. Spectra were acquired using powers ~30x greater than P_1/2, and are aligned using the first moment theorem described by Hyde and Pilbrow [13]. (B) Difference spectrum of the down sweep subtracted from the up sweep.

Figure 7

NARS spectra (up-sweep, normalized to peak) collected at -10°C and 4.1 mW at variable magnetic field sweep rates

Figure 8

NARS spectra (26 μW) collected on the upand down-sweep of the triangle at 65 kG/s and 260 kG/s. In the presence of passage effects, the up- and down-sweep will not be identical.