Abstract
We have used saturation-recovery electron paramagnetic resonance (SR-EPR), a time-resolved saturation transfer EPR technique, to measure directly the microsecond rotational diffusion of spin-labeled proteins. SR-EPR uses an intense microwave pulse to saturate a spin population having narrow distribution of orientations with respect to the magnetic field. The time evolution of the signal is then observed. The signal increases in time as saturation is relieved by spin-lattice relaxation (Tl) as well as by saturation transfer due to spectral diffusion (Tsd), which is a function of rotational diffusion (Tr) and spectral position. In the presence of both events, the recovery is biphasic, with the initial phase related to both Tr and Tl, and the second phase determined only by Tl. We have measured the saturation recoveries of spin-labeled hemoglobin tumbling in media of known viscosities as a function of rotational correlation time (Tr) and pulse duration (tp). The Tr values estimated from the initial phase of recovery were in good agreement with theory. Variation of the pulse time can also be used to determine Tr. For tp less than Tsd, the recoveries were observed to be biphasic, for tp greater than Tsd a single-exponential. T1 values were determined from the recoveries after pulses quenching spectral diffusion or from the second phase of recovery after shorter pulses. These results demonstrate that SR-EPR is applicable to the study of motion of spin-labeled proteins. Its time resolution should provide a significant advantage over steady state techniques, particularly in the case of motional anisotropy or system heterogeneity.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 1195-1202 |
| Number of pages | 8 |
| Journal | Biophysical journal |
| Volume | 50 |
| Issue number | 6 |
| DOIs | |
| State | Published - 1986 |
Bibliographical note
Funding Information:We thank C. Polnaszek and W. Froncisz for many stimulating discus- sions, E. A. Fajer and M. Taylor for technical assistance, S. Flom for the use of his computer program for data analysis, and J. Lipscomb for the use of his computer. This work was supported by grants to D. Thomas from the National Institutes of Health (GM 27906, AM 32961), the American Heart Association, the Muscular Dystrophy Association of America, and the Minnesota Supercomputer Institute; and to J. Hyde from the National Institutes of Health (GM 22923 and RR 01008). P. Fajer was supported by a Muscular Dystrophy Postdoctoral Fellowship. Receivedfor publication S March 1986 and infinalform 3 June 1986.