Sparse labeling PELDOR spectroscopy on multimeric mechanosensitive membrane channels

Pulse 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. homo-multimers) due to distorting multi-spin 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 multi-spin problem.Here, two different appro aches for suppressing multi-spin 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 E. 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 multi-spin 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 analogue.