Pulse electron paramagnetic resonance (EPR) is gaining increasing importance in structural biology. The PELDOR (pulsed electron–electron double resonance) method allows extracting distance information on the nanometer scale. Here, we demonstrate the efficient extraction of distances from multimeric systems such as membrane‐embedded ion channels where data analysis is commonly hindered by multi‐spin effects.

Pulse electron paramagnetic resonance (EPR) is being applied to ever more complex biological systems comprising multiple subunits. Membrane channel proteins are of great interest as pulse EPR reports on functionally significant but distinct conformational states in a native environment without the need for crystallization. Pulse EPR, in the form of pulsed electron-electron double resonance (PELDOR), using site-directed spin labeling, is most commonly employed to accurately determine distances (in the nanometer range) between different regions of the structure. However, PELDOR data analysis is more challenging in systems containing more than two spins (e.g., homomultimers) due to distorting multispin effects. Without suppression of these effects, much of the information contained in PELDOR data cannot be reliably retrieved. Thus, it is of utmost importance for future PELDOR applications in structural biology to develop suitable approaches that can overcome the multispin problem. Here, two different approaches for suppressing multispin effects in PELDOR, sparse labeling of the protein (reducing the labeling efficiency f) and reducing the excitation probability of spins (λ), are compared on two distinct bacterial mechanosensitive channels. For both the pentameric channel of large conductance (MscL) and the heptameric channel of small conductance (MscS) of Escherichia coli, mutants containing a spin label in the cytosolic or the transmembrane region were tested. Data demonstrate that distance distributions can be significantly improved with either approach compared to the standard PELDOR measurement, and confirm that λ < 1/(n−1) is needed to sufficiently suppress multispin effects (with n being the number of spins in the system). A clear advantage of the sparse labeling approach is demonstrated for the cytosolic mutants due to a significantly smaller loss in sensitivity. For the transmembrane mutants, this advantage is less pronounced but still useful for MscS, but performance is inferior for MscL possibly due to structural perturbations by the bulkier diamagnetic spin label analog.

The synthesis of rigid symmetric polyradical model systems with inter-spin distances between 1.4 and 4 nm and their room temperature continuous wave (CW) EPR spectra are reported. Conditions for attachment of the spin-label via esterification have been optimized on the direct synthesis of polyradicals from commercially available polyphenols and the carboxylic acid functionalized nitroxide TPC. A common synthetic protocol utilizing 4-hydroxy-4'-iodobiphenyl as a key building block has been used to synthesize an equilateral biradical and a triradical in only two steps from commercially available starting materials. The first synthesis of a tetraradical based upon an adamantane core bearing six equivalent nitroxide-nitroxide distances is also reported. These systems are very promising candidates for studying multi-spin effects in pulsed EPR distance measurements.

Pulsed electron paramagnetic resonance (EPR) techniques have been found to be efficient tools for the elucidation of structure in complex biological systems as they give access to distances in the nanometre range. These measurements can provide additional structural information such as relative orientations, structural flexibility or aggregation states. A wide variety of model systems for calibration and optimisation of pulsed experiments has been synthesised. Their design is based on mimicking biological systems or materials in specific properties such as the distances themselves and the distance distributions. Here, we review selected approaches to the synthesis of chemical systems bearing two or more spin centres, such as nitroxide or trityl radicals, metal ions or combinations thereof and outline their application in pulsed EPR distance measurements.

A novel polyphenolic backbone, hexakis{4-[(4′-hydroxybiphenyl-4yl)ethynyl]phenyl}benzene, was synthesised using a common synthetic protocol used for the synthesis of similar polyphenolic compounds.