Improved Sensitivity for Long-Distance Measurements in Biomolecules: Five-Pulse Double Electron-Electron Resonance

We describe significantly improved long-distance measurements in biomolecules by use of the new multipulse double electron-electron spin resonance (DEER) illustrated with the example of a five-pulse DEER sequence. In this sequence, an extra pulse at the pump frequency is used compared with standard four-pulse DEER The position of the extra pulse is fixed relative to the three pulses of the detection sequence. This significantly reduces the effect of nuclear spin-diffusion on the electron-spin phase relaxation, thereby enabling longer dipolar evolution times that are required to measure longer distances. Using spin-labeled T4 lysozyme at a concentration less than 50 mu M, as an example, we show that the evolution time increases by a factor of 1.8 in protonated solution and 1.4 in deuterated solution to 8 and 12 mu s, respectively, with the potential to increase them further. This enables a significant increase in the measurable distances, improved distance resolution, or both.