Studying Protein DNA Binding with PELDOR

Abstract

Pulsed Electron-Electron Double Resonance (PELDOR or DEER) is an Electron Paramagnetic Resonance (EPR) method by which distance can be measured very precisely in the range from ∼1-8nm1. As Fluorescence methods, PELDOR usually requires labels but which are considerable smaller and rigid2 making a translation of distance into structure easier.To overcome challenging label synthesis, we introduce the next generation of labels for DNA/RNA which make use of non-covalent binding. We can show that the specificity and strength of binding is large enough to yield high-quality PELDOR data yielding not only distances but also information on orientations and dynamics. This non-covalent labelling concept proves also successful for the study of protein binding and concomitant oligonucleotide bending.Labeling the helicases Hel308 and PcrA, we have been able to resolve the ATP, ADP, and DNA induced conformational switching of these molecular motors.This clearly shows that PELDOR does not only provide mere distances but access to orientations, dynamics and changes between conformational states of large oligonucleotide/protein complexes difficult to access otherwise.1) O. Schiemann, T.F. Prisner Quart. Rev. Biophys. 2007, 40, 1.2) O. Schiemann, P. Cekan, D. Margraf, T.F. Prisner, S.T. Sigurdsson Angew. Chem. 2009, 121, 3342.