Distribution of H$$^\upbeta$$ Hyperfine Couplings in a Tyrosyl Radical Revealed by 263 GHz ENDOR Spectroscopy

Abstract$$^1$$ 1 H ENDOR spectra of tyrosyl radicals (Y$$^\bullet$$ ∙ ) have been the subject of numerous EPR spectroscopic studies due to their importance in biology. Nevertheless, assignment of all internal $$^1$$ 1 H hyperfine couplings has been challenging because of substantial spectral overlap. Recently, using 263 GHz ENDOR in conjunction with statistical analysis, we could identify the signature of the H$$^{\upbeta _2}$$ β 2 coupling in the essential Y$$_{122}$$ 122 radical of Escherichia coli ribonucleotide reductase, and modeled it with a distribution of radical conformations. Here, we demonstrate that this analysis can be extended to the full-width $$^1$$ 1 H ENDOR spectra that contain the larger H$$^{\upbeta _1}$$ β 1 coupling. The H$$^{\upbeta _2}$$ β 2 and H$$^{\upbeta _1}$$ β 1 couplings are related to each other through the ring dihedral and report on the amino acid conformation. The 263 GHz ENDOR data, acquired in batches instead of averaging, and data processing by a new “drift model” allow reconstructing the ENDOR spectra with statistically meaningful confidence intervals and separating them from baseline distortions. Spectral simulations using a distribution of ring dihedral angles confirm the presence of a conformational distribution, consistent with the previous analysis of the H$$^{\upbeta _2}$$ β 2 coupling. The analysis was corroborated by 94 GHz $$^2$$ 2 H ENDOR of deuterated Y$$_{122}^\bullet$$ 122 ∙ . These studies provide a starting point to investigate low populated states of tyrosyl radicals in greater detail.

Mg2+-dependent conformational equilibria in CorA: an integrated view on transport regulation

The CorA family of proteins regulates the homeostasis of divalent metal ions in many bacteria, archaea, and eukaryotic mitochondria, making it an important target in the investigation of the mechanisms of transport and its functional regulation. Although numerous structures of open and closed channels are now available for the CorA family, the mechanism of the transport regulation remains elusive. Here, we investigated the conformational distribution and associated dynamic behaviour of the pentameric Mg2+ channel CorA at room temperature using small-angle neutron scattering (SANS) in combination with molecular dynamics (MD) simulations and solid-state nuclear magnetic resonance spectroscopy (NMR). We find that neither the Mg2+-bound closed structure nor the Mg2+-free open forms are sufficient to explain the average conformation of CorA. Our data support the presence of conformational equilibria between multiple states, and we further find a variation in the behaviour of the backbone dynamics with and without Mg2+. We propose that CorA must be in a dynamic equilibrium between different non-conducting states, both symmetric and asymmetric, regardless of bound Mg2+ but that conducting states become more populated in Mg2+-free conditions. These properties are regulated by backbone dynamics and are key to understanding the functional regulation of CorA.

Modifying conformational distribution of chiral tetrahydro-2-furoic acid through its interaction with water: a rotational spectroscopic and theoretical investigation

The preferred conformations and conformational cooling paths of tetrahydro-2-furoic acid–water are established and the “lubrification” effects of water revealed.

TD-DFT and Experimental Methods for Unraveling the Energy Distribution of Charge-Transfer Triplet/Singlet States of a TADF Molecule in a Frozen Matrix

Reverse intersystem crossing (RISC) rate of a thermally activated delayed fluorescence (TADF) molecule is sensitive to the energy alignment of singlet charge-transfer state (¹CT), triplet charge-transfer state (³CT), and locally excited triplet state (³LE). However, the energy distribution of the charge-transfer states originating from the conformational distribution of TADF molecules in a solid matrix inevitably generated during the preparation of a solid sample due to the rotatable donor-acceptor linkage is rarely considered. Moreover, the investigation of the energy distribution of the ³CT state is both theoretically and experimentally difficult due to the triplet instabilities of time-dependent density functional (TD-DFT) calculations and difficulties in phosphorescence measurements, respectively. As a result, the relation between conformational distribution, configurations of excited state transition orbitals, and excited state energies/dynamics have not been clearly explained. In this work, we determined the energy distribution of CT states of the TADF emitter TPSA in frozen toluene at 77 K by the measurement of time-resolved spectra in the full time range (1 ns ~ 30 s) of emission including prompt fluorescence, TADF, ³CT phosphorescence, and ³LE phosphorescence. We obtained the energy band of CT states where ¹CT and ³CT states are distributed in the range of 2.85-3.00 eV and 2.64-2.96 eV, respectively. We tested various global hybrid and long-range corrected functionals for the TD-DFT calculation of ³CT energy of TPSA and found that only the M11 functional shows consistent results without triplet instability. We performed TD-DFT with the M11* functional optimized for robust dihedral angle scan of ³CT states without triplet instability and reproduced the energy band structure obtained from the experiment. Through TD-DFT and experimental investigations, it is estimated that the dihedral angle of donor-acceptor (θ_D-A) and acceptor-linker (θ_A) of TPSA in frozen toluene lie within the range of 70°≤θ_D-A≤90° and 0°≤θ_A≤30° respectively. Our results show that the dihedral angle distribution must be considered for further investigation of the photophysics of TADF molecules and the development of stable and efficient TADF emitters.

TD-DFT and Experimental Methods for Unraveling the Energy Distribution of Charge-Transfer Triplet/Singlet States of a TADF Molecule in a Frozen Matrix

Reverse intersystem crossing (RISC) rate of a thermally activated delayed fluorescence (TADF) molecule is sensitive to the energy alignment of singlet charge-transfer state (¹CT), triplet charge-transfer state (³CT), and locally excited triplet state (³LE). However, the energy distribution of the charge-transfer states originating from the conformational distribution of TADF molecules in a solid matrix inevitably generated during the preparation of a solid sample due to the rotatable donor-acceptor linkage is rarely considered. Moreover, the investigation of the energy distribution of the ³CT state is both theoretically and experimentally difficult due to the triplet instabilities of time-dependent density functional (TD-DFT) calculations and difficulties in phosphorescence measurements, respectively. As a result, the relation between conformational distribution, configurations of excited state transition orbitals, and excited state energies/dynamics have not been clearly explained. In this work, we determined the energy distribution of CT states of the TADF emitter TPSA in frozen toluene at 77 K by the measurement of time-resolved spectra in the full time range (1 ns ~ 30 s) of emission including prompt fluorescence, TADF, ³CT phosphorescence, and ³LE phosphorescence. We obtained the energy band of CT states where ¹CT and ³CT states are distributed in the range of 2.85-3.00 eV and 2.64-2.96 eV, respectively. We tested various global hybrid and long-range corrected functionals for the TD-DFT calculation of ³CT energy of TPSA and found that only the M11 functional shows consistent results without triplet instability. We performed TD-DFT with the M11* functional optimized for robust dihedral angle scan of ³CT states without triplet instability and reproduced the energy band structure obtained from the experiment. Through TD-DFT and experimental investigations, it is estimated that the dihedral angle of donor-acceptor (θ_D-A) and acceptor-linker (θ_A) of TPSA in frozen toluene lie within the range of 70°≤θ_D-A≤90° and 0°≤θ_A≤30° respectively. Our results show that the dihedral angle distribution must be considered for further investigation of the photophysics of TADF molecules and the development of stable and efficient TADF emitters.