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. Author manuscript; available in PMC: 2017 Jan 9.
Published in final edited form as: Methods Enzymol. 2015 Aug 29;564:349–387. doi: 10.1016/bs.mie.2015.07.026

Figure 3. Site-directed spin labeling and correlation of the EPR spectrum with local structure.

Figure 3

(A) Targeted cysteine mutagenesis introduces a sulfhydryl moiety for the attachment of a nitroxide spin label, such as MTSSL. Rotational isomerization of MTSSL predominantly around the bonds highlighted in gray is reflected in the EPR spectral lineshape. (B) The degree of rotational freedom of the label is determined by the local packing environment. Fast rotational correlation times (~1ns) correspond to spin labels attached to surface-exposed sites. Tertiary contact interactions or buried sites that restrict spin label motion reduce the rate and amplitude of isomerization leading to broadening of the lineshape. The dashed line emphasizes the progressive appearance of a slow motion component associated with restricted rotation.