Gadolinium (III) Spin Labels for High-Sensitivity Distance Measurements in Transmembrane Helices

摘要

Distance determination, by pulse EPR techniques, between two spin labels attached to biomolecules has become an attractive methodology to probe conformations and assemblies of biomolecules in frozen solutions. Among these techniques, double electron-electron resonance (DEER or PELDOR), which can access distances in the range of 1.7 to 8 nm, is highly popular, and the most widely used spin labels are nitroxide radicals. Membrane proteins in their natural environment are of particular interest for DEER applications, since those pose a considerable challenge for X-ray crystallography and NMR spectroscopy. DEER studies of peptides and proteins in either reconstituted or model membranes are considerably more challenging than those in solution, because the high local concentration of the spins in the membrane decreases the phase memory time and, therefore, sensitivity. Most DEER measurements on nitroxide-labeled biomolecules are carried out at X-band frequencies (9.5 GHz, 0.35 T), and recently such measurements were demonstrated in frozen cells. A major difficulty of such measurements is the reduction of nitroxides in the cell, which severely limits the scope of such exciting developments.