Calmodulin (CaM) is a relatively small protein abundant in eukaryotic cells that plays a pivotal role in sensing and responding to modulation of calcium levels (Figure ). As such, CaM regulates the activity of a large number of important protein targets, and therefore plays a significant role in various aspects of human health. As a biologically important, thoroughly studied and structurally well-understood protein, we identified CaM as a suitable protein to benchmark the use of iodoacetamide 12 for spin labelling. More specifically, we chose to employ the complex formed by Ca 2+ -bound holo-CaM and the synthetic peptide M13 via a GG linker (CaM/M13), which mimics the CaM-binding domain of rabbit skeletal muscle myosin light chain kinase, as our model protein (Figure ). As the native CaM/M13 amino acid sequence lacks cysteine residues suitable for labelling with iodoacetamide spin labels, a double mutant with cysteines at residues 34 and 146 (highlighted yellow residues in Figure ) was produced by mutagenesis (see the Supplementary Information for details). Spin labelling of the double cysteine variant of CaM/M13 was performed using 50 µM protein and 500 µM nitroxide 12 in HEPES buffer (40 mM, pH 7.5) and NaCl solution (150 mM). Due to the light sensitive nature of iodoacetamides, the mixture was incubated in darkness at 4 °C for 16 h. This protocol yielded a 3:10 ratio of singly-and doubly-labelled protein, with a notable 77% efficiency in double labelling according to analysis by ESI- MS (see the Supplementary Information, Figure ). The doubly-labelled protein was subsequently isolated after functionalising any unreacted free cysteines with biotin and purification using streptavidin-coated beads (see the Supplementary Information, Figure ). Spirocyclic pyrrolinyl nitroxide spin labels have been synthesized previously, but to the best of our knowledge, this is the first example of fivemembered nitroxides with spirocyclohexyl moieties being used in protein spin labelling. To assess the stability of the spin label in a reducing environment, we recorded X-band continuous wave (CW) EPR spectra of the doubly-labelled protein (250 µM in HEPES buffer, pH 7.5) in the presence of a 32-fold (16-fold per label) excess of the reducing agent sodium ascorbate (Figure ). The data shows a 50% decrease in the intensity of the CW spectrum over approximately 20 minutes, and a 66% decrease after 40 minutes. These measurements confirm that the EPR signal of spin label 12 when attached to the protein remains sufficiently strong for long enough to permit spectroscopic measurements, possibly even in challenging reducing media such as the intracellular environment.