Distance distribution information obtained by pulsed dipolar EPR spectroscopy provides an important contribution to many studies in structural biology. Increasingly, such information is used in integrative structural modeling, where it delivers unique restraints on the width of conformational ensembles. In order to ensure reliability of the structural models and of biological conclusions, we herein define quality standards for sample preparation and characterization, for measurements of distributed dipole–dipole couplings between paramagnetic labels, for conversion of the primary time-domain data into distance distributions, for interpreting these distributions, and for reporting results. These guidelines are substantiated by a multi-laboratory benchmark study and by analysis of data sets with known distance distribution ground truth. The study and the guidelines focus on proteins labeled with nitroxides and on double electron–electron resonance (DEER aka PELDOR) measurements and provide suggestions on how to proceed analogously in other cases.

Photosensitizers with high energy, long lasting charge-transfer states are important components in systems designed for solar energy conversion by multistep electron transfer. Here, we show that in a push–pull type, μ-oxo-bridged porphyrin heterodimer composed of octaethylporphyrinatoaluminum(III) and octaethylporphyrinatophosphorus(V), the strong excitonic coupling between the porphyrins and the different electron withdrawing abilities of Al(III) and P(V) promote the formation of a high energy CT state. Using, an array of optical and magnetic resonance spectroscopic methods along with theoretical calculations, we demonstrate photodynamics of the heterodimer that involves the initial formation of a singlet CT which relaxes to a triplet CT state with a lifetime of ∼130 ps. The high-energy triplet CT state (³CT = 1.68 eV) lasts for nearly 105 μs prior to relaxing to the ground state.

We present a new technique, light-induced triplet–triplet electron resonance spectroscopy (LITTER), which measures the dipolar interaction between two photoexcited triplet states, enabling both the distance and angular distributions between the two triplet moieties to be determined on a nanometer scale. This is demonstrated for a model bis-porphyrin peptide that renders dipolar traces with strong orientation selection effects. Using simulations and density functional theory calculations, we extract distance distributions and relative orientations of the porphyrin moieties, allowing the dominant conformation of the peptide in a frozen solution to be identified. LITTER removes the requirement of current light-induced electron spin resonance pulse dipolar spectroscopy techniques to have a permanent paramagnetic moiety, becoming more suitable for in-cell applications and facilitating access to distance determination in unmodified macromolecular systems containing photoexcitable moieties. LITTER also has the potential to enable direct comparison with Förster resonance energy transfer and combination with microscopy inside cells.

Carotenoid molecules are essential for the life of photosynthetic organisms in that they protect the cell from the photo-oxidative damage induced by light-stress conditions. One of the photo-protective mechanisms involves triplet–triplet energy transfer from the chlorophyll molecules to the carotenoids: a process that is strongly dependent on the electronic properties of the triplet states involved. Here, we obtain a clear description of the triplet state of lutein in LHCII from higher plants for the first time by density functional theory (DFT) calculations. DFT predictions have been validated by comparison with hyperfine couplings obtained with pulsed-ENDOR spectroscopy. Knowledge of the spin density distribution, the frontier orbitals and orbital excitations forms a basis for discussing the requirements for an efficient triplet–triplet energy transfer. The results obtained for the lutein in LHCII are compared with those of the highly-substituted carotenoid peridinin in PCP from Amphidinium carterae [Di Valentin et al., Biochim. Biophys. Acta, 2008, 1777, 295–307]. The presence of substituents in the peridinin molecule does not alter significantly the triplet state electronic structure compared to lutein. Despite the unusual spectroscopic behaviour of the peridinin excited singlet state, lutein and peridinin have similar triplet state properties. In both molecules the unpaired spins are delocalized uniformly over the whole π-conjugated system in an alternating even–odd pattern.