Site directed spin labeling (SDSL) and quantifying membrane protein dynamics

In SDSL, a radical-containing nitroxide spin label is site specifically introduced into a protein (e.g., selectively reacted with the free sulfhydryl of a single cysteine). The most commonly used spin label is a methanethiosulfonate derivative, designated R1, which is attached to the protein via a disulfide bond; this is the label used most often in the lab. The resulting EPR spectrum reflects the overall rotational motions of the nitroxide ring on a nanosecond timescale (at X-band; 9.5 GHz). The dynamic modes that contribute to the motion of the nitroxide in membrane proteins are the side chain and backbone motions. The Columbus laboratory aims to characterize the structure and quantify the dynamics of nitroxides on membrane proteins in order to understand the resulting EPR lineshape.
–Brett Kroncke and Justin Kim

Improving precision and accuracy of paramagnetic relaxation enhancement (PRE) distance restraints
Using the spin label dynamics and structural information gained from the above studies, the Columbus laboratory aims to improve the precision and accuracy of paramagnetic relaxation enhancement (PRE; due to the presence of the spin label) derived restraints for nuclear magnetic resonance (NMR) structure calculations. To date, only eight polytopic membrane protein structures have been determined with NMR. A major hurdle is the constraint of working with dueterated samples, a requisite for large protein complexes. This restriction limits the number of obtainable nuclear overhauser effect (NOE) distance restraints, especially for α-helical membrane proteins. Alternatively, PRE distance restraints, due to the distance-dependent enhanced relaxation of protons by a paramagnetic center–such as the spin label R1–, are well suited to provide long-range structure information on membrane proteins.
–Brett Kroncke, Dan Fox, and Justin Kim