Saturation transfer EPR spectroscopy on spin-labeled muscle fibers using a loop-gap resonator

D. D. Thomas, C. H. Wendt, W. Francisz, J. S. Hyde

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Previously, saturation transfer (ST-EPR) studies of biomolecular dynamics have involved the use of a resonant cavity and the V'2 display (absorption, second harmonic, out of phase). In the present study, we replaced the resonant cavity with a loop-gap resonator and used the U'1 display (dispersion, first harmonic, out of phase) to study spin-labeled muscle fibers. The new resonator and display showed several advantages over those previously used. It produced virtually noiseless U'1 spectra on a 0.4 microliter sample using a 4 min scan; previous U'1 experiments on spin-labeled muscle, using a conventional rectangular cavity, resulted in an unacceptably low signal-to-noise ratio. The high filling factor of the resonator facilitated the study of these extremely small fiber bundles and permitted high microwave field intensities to be achieved at much lower incident microwave power levels, thus greatly enhancing the signal-to-noise ratio in U'1 experiments. This reduction in the noise level made it possible to benefit from the other advantages of U'1 over V'2, such as stronger signals, simpler line shapes, and simpler data analysis. For these muscle fiber samples, the resulting sensitivity (signal/noise/sample volume) of the U'1 signals was greater than 100 times that of V'2 signals obtained in a conventional cavity. Another advantage of the U'1 display is that signals from weakly immobilized probes, i.e., probes that have nanosecond rotational mobility relative to the labeled protein (myosin), are greatly suppressed relative to strongly immobilized probes. This reduces the ambiguity of spectral analysis, and eliminates the need for chemical treatments [e.g., using K3Fe(CN)6] that were previously required in muscle fibers and other systems. Further suppression of this weakly immobilized component was achieved in U'1 spectra by increasing the microwave power and decreasing the field modulation frequency.

Original languageEnglish (US)
Pages (from-to)131-135
Number of pages5
JournalBiophysical journal
Issue number1
StatePublished - 1983

Bibliographical note

Funding Information:
We thank Vincent Barnett, Roger Cooke, and Akihiro Kusumi for advice and assistance. This work was supported by grants to Dr. Thomas from the National Institutes of Health (GM 27906), the American Heart Association (80 850), the National Science Foundation (PCM 8004612), and the Muscular Dystrophy Association of America. Dr. Thomas was supported by a Research Career Development Award from the National Institutes of Health (AM 00851), and is currently supported by an Established Investigatorship from the American Heart Association. This work was supported by grants to Dr. Hyde from the National Institutes of Health (GM-27665 and RR-01008) and the National Science Foundation (PCM-7823206 and PCM-8118976).


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