"Store-bought is fine": Sensitivity considerations using shaped pulses for DEER measurements on Cu(II) labels.

ABSTRACT

The narrow excitation bandwidth of monochromic pulses is a sensitivity limitation for pulsed dipolar spectroscopy on Cu(II)-based measurements. In response, frequency-swept pulses with large excitation bandwidths have been adopted to probe a greater range of the EPR spectrum. However, much of the work utilizing frequency-swept pulses in Cu(II) distance measurements has been carried out on home-built spectrometers and equipment. Herein, we carry out systematic Cu(II) based distance measurements to demonstrate the capability of chirp pulses on commercial instrumentation. More importantly we delineate sensitivity considerations under acquisition schemes that are necessary for robust distance measurements using Cu(II) labels for proteins. We show that a 200 MHz sweeping bandwidth chirp pulse can improve the sensitivity of long-range distance measurements by factors of three to four. The sensitivity of short-range distances only increases slightly due to special considerations for the chirp pulse duration relative to the period length of the modulated dipolar signal. Enhancements in sensitivity also dramatically reduce measurement collection times enabling rapid collection of orientationally averaged Cu(II) distance measurements in under two hours.