High Power Pulsed ESR at 95GHz

Two-dimensional time-domain studies provide exceptional sensitivity to ordering and dynamics, as compared to cw-ESR, in the study of the dynamic structure of membranes and biopolymers. 2D-ELDOR and related 2D techniques also permit the study of much slower processes than cw-ESR at a comparable frequency, which is an important consideration for the complex dynamics of biological systems. This capability can be greatly enhanced when supplemented with high-field/high-frequency (HFHF) ESR. For nitroxide spin-labels, HFHF-ESR provides much better orientational resolution, which greatly aids in determining the correct dynamical model from the spectrum. It also provides greater sensitivity to the faster dynamics than ESR at conventional frequencies. The combination of all these features in a pulsed HFHF-ESR spectrometer would yield enhanced capabilities for studies of dynamic structure in biological systems. We have just recently completed the construction of a high-power pulsed ESR spectrometer operating at 95GHz. Although several laboratories have performed pulsed ESR at frequencies in the range of 95 to 150GHz, all the past experiments have been at low powers, corresponding to π/2 pulses of ca. 50–100ns. This is completely unsatisfactory for work on molecular dynamics of biomolecules in fluid media, at or near physiological temperatures, for which the homogeneous T₂'s can be as short as several nanoseconds. Our new spectrometer has been tested on motionally-narrowed nitroxide spectra, from which we have been able to obtain FID's and 2D-COSY spectra with π/2 pulses as short as 4ns in favorable cases. We have recently succeeded in performing 2D-ELDOR experiments on model systems. We show representative 2D-ELDOR spectra of the nitroxide probe TEMPO in a hydrocarbon solvent that demonstrate the development of spin exchange cross-peaks (due to collisions of nitroxide molecules) as a function of increasing mixing time. This new spectrometer has many of the features required to study samples of biological interest such as spin-labeled proteins and membranes. It is a unique achievement with no other comparable pulsed ESR spectrometer elsewhere.