Benchmark Test and Guidelines for DEER/PELDOR Experiments on Nitroxide-Labeled Biomolecules.

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.

Spirocyclic pyrrolidinyl nitroxides with exo-methylene substituents.

Nitroxides are stable organic radicals with exceptionally long lifetimes, which render them uniquely suitable as observable probes or polarising agents for spectroscopic investigation of biomolecular structure and dynamics. Radical-based probes for biological applications are ideally characterized by both robustness towards reductive degradation and beneficial electron spin relaxation parameters. These properties are largely influenced by the molecular structure of the nitroxide scaffold, and also by the conformations it prefers to adopt. In this study we present the synthesis of the first nitroxides based on a spirocyclic pyrrolidine scaffold with an exocyclic methylene substituent. The conformations adopted by these nitroxides were evaluated by X-ray crystallography, both with single nitroxide crystals and by inclusion of nitroxides in a microporous crystalline sponge. The kinetic and thermodynamic stability of the new nitroxides towards reduction was investigated by electron paramagnetic resonance (EPR) spectroscopy and cyclic voltammetry (CV). In combination with EPR measurements of electron spin relaxation properties, these results suggest that this new family of nitroxides can provide access to multifunctionalized probes and polarising agents suitable for use in biological environments at elevated temperatures.