Non-canonical amino acids for site-directed spin labeling of membrane proteins

Membrane proteins remain challenging targets for conventional structural biology techniques because they need to reside within complex hydrophobic lipid environments to maintain proper structure and function. Magnetic resonance combined with site-directed spin labeling is an alternative method that provides atomic-level structural and dynamical information from effects introduced by an electron- or nuclear-based spin label. With the advent of bioorthogonal click chemistries and genetically engineered non-canonical amino acids (ncAAs), options for linking spin probes to biomolecules have substantially broadened outside the conventional cysteine-based labeling scheme. Here, we highlight current strategies to spin-label membrane proteins through ncAAs for nuclear and electron paramagnetic resonance applications. Such advances are critical for developing bioorthogonal spin labeling schemes to achieve in-cell labeling and in-cell measurements of membrane protein conformational dynamics.