dHis-Cu(II) based EPR distances at W-band: navigating the challenges of excitation

The measurement of nanometer range distance constraints by pulsed dipolar EPR spectroscopy has become an invaluable tool for integrative biophysics. Combining rigid spin labels, such as Cu(II)-NTA, with high frequency EPR is attractive to measure structural information with high precision. Such measurements are, however, challenged by limited excitation bandwidth which can lead to complications in both data acquisition and analysis. Herein, we examine the distance measurements on proteins labeled with Cu(II) at W-band (ca. 95 GHz) frequencies. The large anisotropy of the g-tensor leads to a spectral width of about ten GHz. Thus, the acquisition of an orientation independent dipolar trace becomes non-trivial with pulses of three orders of magnitude smaller bandwidth. We show using a recently developed computational approach that acquisition at only four field positions is sufficient to yield orientation averaged distance distributions. This acquisition relies on excitation of only ∼0.43% of spins in total. The computational approach is validated on data from two samples of doubly labeled GB1 protein. We anticipate that this conceptual methodology will be of use for a wide variety of high field pulsed EPR measurements.