MTSL

MTSL is attached to proteins by reaction with thiol groups. The reaction exploits standard reactivity of thiosulfate esters. Methanesulfinate (CH₃SO₂⁻) is the leaving group:

RSO₂S-nitroxide + protein-SH → protein-S-S-nitroxide + RSO₂H

The heterodisulfide bond to the cysteine residue is robust, enabling site-directed spin labelling.^[2][3] The MTSL moiety will add 184.3 daltons to the mass of the protein or peptide to which it is attached. The cysteine can be introduced using site-directed mutagenesis, and hence most positions in a protein can be labelled.

In Nuclear magnetic resonance the introduction of the paramagnetic group increases the relaxation rate of nearby nuclei.^[1] Its presence can be detected as peak broadening and loss of intensity in peaks corresponding to nearby nuclei. Hence proximity can be inferred for all nuclei, that are affected. A major advantage of this method over traditional methods for obtaining distance restraints in protein NMR is the increased length, as paramagnetic relaxation enhancement can detect distances up to 25 Å (2.5 nm) as opposed to about 6 Å (0.6 nm) using the nuclear Overhauser effect. Spin labelling with MTSL is frequently used in investigation of residual structure in intrinsically unstructured proteins.