High-field/High-frequency EPR Spectroscopy in Protein Research: Principles and Examples

Abstract

During the last decades, the combined efforts of biologists, chemists, and physicists in developing high-field/high-frequency EPR techniques and applying them to functional proteins have demonstrated that this type of magnetic resonance spectroscopy is particularly powerful for characterizing the structure and dynamics of stable and transient states of proteins in action on biologically relevant time scales ranging from nanoseconds to hours. The review article describes how high-field EPR methodology, in conjunction with site-specific isotope and spin-labeling strategies, is capable of providing new insights into fundamental biological processes. Specifically, we discuss the theoretical and instrumental background of continuous-wave and pulse high-field EPR and the multiple-resonance extensions EDNMR, ENDOR, TRIPLE, ESEEM, PELDOR, and RIDME. Some emphasis is placed on a balanced description of both the historical spadework and the achieved performance of advanced EPR at 95 GHz and 360 GHz. This culminates in a coherent treatment of state-of-the-art research of high-field EPR in terms of both instrumentation development and application to representative protein complexes such as cofactor binding sites in photosynthesis.

Conditions for EPR detection of chirality-induced spin selectivity in spin-polarized radical pairs in isotropic solution

“…Continuous-wave, time-resolved EPR spectra were simulated using an approach 2,4,26,27 previously employed to interpret spectra of light-induced spin-correlated radical pairs (SCRPs) in photosynthetic reaction centre proteins 2,4,26,[28][29][30][31][32] and in a variety of model compounds. 6,[33][34][35] Briefly, the amplitudes of individual EPR transitions are calculated as the products of their polarizations and their transition probabilities; spin coherences are ignored by virtue of fast dephasing, interference effects, and experimental timing; hyperfine interactions are assumed to give rise to isotropic inhomogeneous line-broadening; spin-lattice relaxation is assumed to be too slow to affect the observed polarizations; and molecular motion is ignored.…”

A design of resonant cavity with an improved coupling-adjusting mechanism for the W-band EPR spectrometer

“…The characteristic shape of the spectrum is due to the anisotropy of g factor and hyperfine splitting. [12] The g zz peak splits into three because of the A zz hyperfine-tensor component. They are corresponding to the three peaks observed in Fig.…”

“…HF-EPR provides higher spectral resolution, stronger orientational selectivity and less requirement on sample volume, and opens the path to understanding the fast motional dynamics of molecules. [12] Some analogous technologies, such as nuclear magnetic resonance (NMR) [13] and muon spin rotation/relaxation/resonance (µSR), [14] also benefit from highfrequency high-magnetic field. [15,16] A microwave (mw) resonator is one of the most significant parts in an EPR spectrometer since it greatly influences the performance of the spectrometer.…”

Conversion of a Single-Frequency X-Band EPR Spectrometer into a Broadband Multi-Frequency 0.1–18 GHz Instrument for Analysis of Complex Molecular Spin Hamiltonians

“…Collection of data at multiple frequencies, and their subsequent global analysis, increases our chances for a meaningful spectral interpretation in terms of a unique spin Hamiltonian. A common application of this philosophy is multiple high-frequency EPR for the resolution of g matrices with small anisotropy and for the detection of systems with zero-field splitting significantly greater than the standard X-band quantum of ca 0.3 cm −1 [ 1 , 2 ]. A similar, though less common, approach at lower frequencies (of order X-band and below) is indicated for resolution and interpretation of complex patterns of hyperfine and/or dipolar interaction.…”