Abstract
Site-specific attachment of paramagnetic spin labels to biomolecules causes distance-dependent line-broadening effects, which can be exploited to study the structure and dynamics of these molecules in solution. This protocol describes how to attach nitroxide spin labels to proteins and how to collect and analyze NMR data using these labeled samples. We also explain how to derive distance restraints for paramagnetic relaxation enhancement nuclear magnetic resonance (PRE-NMR) studies.