Alexey V. Bogdanov

1 Coherent electron-spin dynamics has been shown to modulate flavoprotein fluorescence in vivo , but not the chemistry by which a protein signals. Here we show that a magnetic field modulates formation of the covalent FMN-cysteine photoadduct that constitutes the signaling state of wild-type Avena sativa LOV2 (AsLOV2), via a spin-selective radical-pair reaction. AsLOV2 was thought magnetically inactive, but a fluorescence magnetic field effect (MFE) emerges near 100 mT and reaches 7% by 1300 mT. Tracking the photoadduct by UV-vis absorption, we find that 650 mT raises the signaling state population and shortens the product formation time constant by 6.7 ± 0.6%. The short FMN-cysteine separation produces a large dipolar coupling that blocks hyperfine-driven triplet-singlet interconversion at low field; the unusually large flavin-cysteinyl radical g -factor difference overcomes it at high field. The same proximity enables covalent bond formation of radical pairs in a singlet state. This arrangement is canonical in LOV domains, so magnetically enhanced signaling likely extends across natural and engineered photoreceptors.

The zero-field splitting (ZFS) of Gd(III) complexes is central to magnetic resonance applications, influencing nuclear relaxation in MRI and NMR, electron spin relaxation in EPR, and the performance of Gd-based spin labels for structural biology applications. However, determining the molecular-frame orientation of the ZFS tensor is experimentally and computationally demanding for Gd(III) chelate complexes in frozen solution, where structural and dynamic heterogeneity complicates orientation determination. Here, we introduce an experimental strategy that enables direct determination of the ZFS tensor orientation in Gd(III) complexes using 19F and 1H orientation-selective (OS) electron–nuclear double resonance (ENDOR). For the commonly used spin labels Gd-DO3A and Gd-PyMTA, we determined the molecular-frame orientation of the ZFS, necessary for quantitative analysis of the Gd(III) anisotropy. This allowed for a dual-mode analysis of Gd–F ENDOR spectra, where electron–nuclear distances can be accurately extracted from measurements at the Gd(III) central transition, while the Gd–F vector orientation is determined using OS-ENDOR on high-|mS| transitions. Accordingly, we determined the location of Gd-DO3A labels in two 19F-containing proteins and showed that utilizing existing rotamer libraries substantially overestimates the label’s conformational distributions. The experimentally determined ZFS orientations are further compared to theoretical predictions of a simple molecular modeling calculation based on the electric-field-gradient tensor. Our results provide a platform for improved structure determination with Gd-based labels and offer valuable benchmarks for improving quantum-chemical predictions of ZFS tensors.

Dynamic nuclear polarization (DNP) overcomes the inherent low sensitivity of solid-state NMR, but its advancement hinges on rational design of polarizing agents (PAs). Recent conflicting reports on tethering or mixing one trityl with two nitroxides motivated us to develop two series of multiradical-based PAs, named as MRPols and cMRPols, which feature one trityl OX063 tethered to 1-3 TEMPO or spirocyclohexanoyl nitroxides (SCHN), respectively. DNP results on [¹³C-¹⁵N] proline at 18.8 T showed that the linkage of two nitroxides into PAs (i.e., MRPol-2 and cMRPol-2) substantially shortened the polarization buildup times (T_B) compared to the single nitroxide-containing biradicals MRPol-1 and cMRPol-1. Notably, the SCHN-based triradical cMRPol-2 with a short T_B (3.24 s) and excellent ε_on/off value (160) provided 65% and 35% improvements in DNP efficiency (ε_on/off/√T_B) over the biradical cMRPol-1 and the benchmark PA SNAPol-1, respectively. The correlation of the excellent DNP performance of cMRPol-2 with the physicochemical properties of the two radical moieties (i.e., electron relaxation and hydrophilicity), as well as trityl-nitroxide and nitroxide-nitroxide interactions, was investigated. Furthermore, the biological application potential of cMRPol-2 was also demonstrated by its high DNP efficiency for cell membrane fractions, remarkable bioreduction resistance, and negligible protein binding. To the best of our knowledge, this work represents the first successful optimization of the DNP efficiency of PAs by balancing T_B and ε_on/off at high magnetic fields (e.g., 18.8 T), and the hetero triradical design will be an applicable strategy for improving DNP efficiency.

Electron-nuclear double resonance (ENDOR) is a powerful tool for determining the spatial and electronic structure of paramagnetic systems. It often suffers from a limited signal-to-noise ratio (SNR), particularly for small hyperfine couplings, which correspond to long-range electron-nuclear distances. We implement a Hadamard frequency multiplexing strategy to enhance the sensitivity in frequency-domain ENDOR spectroscopy. This method makes use of simultaneous or sequential excitation of multiple nuclear frequencies within a single pulse sequence, with spectral reconstruction via a Hadamard transform. Using fluorine ENDOR of fluorinated small molecules and spin-labeled proteins, we demonstrate up to 2-fold improvement in SNR. Approaches that mitigate the limitations of radiofrequency power and relaxation effects are presented, making this strategy useful for both organic radicals and paramagnetic metal complexes. Beyond ENDOR, Hadamard encoded acquisition also improves electron double resonance detected NMR sensitivity, suggesting broad applicability across EPR methods that rely on frequency sweeps.

Dynamic nuclear polarization (DNP) is a powerful route for overcoming the inherent sensitivity limitation of solid-state nuclear magnetic resonance (ssNMR) spectroscopy by transferring high electron spin polarization to surrounding nuclear spins. Cross-effect (CE) DNP is the most efficient mechanism in solids. CE requires several conditions to be met, primarily the presence of two coupled electron spins with resonance frequencies separated by the nuclear Larmor frequency. This condition is typically achieved through the presence of large anisotropic spin interactions, which shift the transition frequencies of the two coupled electron spins with respect to each other. Here we present an alternative approach, where the CE condition is met via isotropic interactions. This is advantageous as it makes CE independent of the sample orientation, thus making the enhancements independent of the MAS frequency and enabling the use of fast relaxing polarizing agents. We demonstrate the feasibility of the approach in experiments and simulations for Mn(II) dopants as polarizing agents, making use of the isotropic hyperfine interactions with its ⁵⁵Mn nuclear spin to achieve the required frequency difference.

Time-domain electron-nuclear double resonance (ENDOR) was employed to determine the nuclear decoherence rate (1/T_2n) of fluorine nuclei in four model Gd(III) chelates of varying structures, over the temperature range 3.7-10 K. These complexes are derivatives of Gd(III) spin labels used in ¹⁹F-ENDOR distance measurements, which emerged as an efficient method for measuring short-range distances in biomolecules. It was found that the ¹⁹F decoherence originates primarily from electron spin flips, following the relationship T_2n ≈ 2T_1e, where T_1e is the Gd(III) spin-lattice relaxation time. The obtained ¹⁹F decoherence rates (1-8 kHz) indicate that the intrinsic ¹⁹F-ENDOR line widths are small relative to other broadening contributions, and therefore do not significantly limit distance resolution of ¹⁹F ENDOR. Proton decoherence measurements were carried out for comparison, revealing two ¹H populations with different relaxation rates; the faster component was sensitive to the matrix protonation and attributed to dipolar flip-flops among weakly coupled protons, a mechanism absent for ¹⁹F. These results elucidate key factors affecting ENDOR resolution and provide new insights into the nuclear spin relaxation mechanism in paramagnetic systems.

Pulse-dipolar electron paramagnetic resonance (PD-EPR) has emerged as an effective tool in structural biology, enabling distance measurements between spin labels attached to biomolecules. The sensitivity and the accessible distance range of these measurements are governed by the phase memory time (T_m) of the spin labels. Understanding the decoherence mechanisms affecting T_m is crucial for optimizing sample preparation and spin-label design. This study investigates the phase relaxation behavior of two Gd(III) spin-label complexes, Gd-PyMTA and Gd-TPMTA, with various degrees of deuteration. These two complexes have significantly different zero-field splitting (ZFS) parameters. Hahn echo decay and dynamical decoupling (DD) measurements were performed at W-band (95 GHz) in deuterated solvents (D₂O/glycerol-d₈), both for the free complexes and when conjugated to proteins. The impact of temperature, concentration, and field position within the EPR spectrum on T_m was examined. Results indicate that protons within 5 Å of the Gd(III) ion do not contribute to nuclear spin diffusion (NSD), and protein deuteration offers minimal enhancement in T_m. The dominant phase relaxation mechanisms identified at low concentrations were direct spin-lattice relaxation (T₁) and transient ZFS fluctuations (tZFS). Dynamical decoupling (DD) measurements, using the Carr-Purcel sequence with ~140 refocusing pulses, resolved the presence of two populations: one with a long phase relaxation time, T_m,s, and the other with a short one, T_m,f. The dominating mechanism for the slowly relaxing population is direct-T₁. T_m,s showed no concentration dependence and was longer by a factor of about 2 from T_m for both complexes. We tentatively assign the increase in T_m,s to full suppression of the residual indirect T₁-induced NSD mechanism. For the fast relaxing population, T_m,f is shorter for Gd-TPMTA; therefore, we assign it to populations for which the tZFS mechanism dominates. Because of the relatively short T₁ and the contribution of tZFS mechanism, protein deuteration does not significantly affect T_m.

¹⁹ F ENDOR is used to monitor the exact position of fluorinated guests in Gd-capped cyclodextrin cavitands.