The purpose of this application is to obtain an electron paramagnetic resonance (EPR) spectrometer and accessories for use as a shared instrument by a group of investigators at the University of Minnesota. These researchers use nitroxide spin labels to study molecular dynamics under physiological conditions in cells or in subcellular model systems. This spectrometer will be used primarily to continue and expand NIH-funded research previously carried out using either a 17-year-old spectrometer that has become unusable or a 10-year-old spectrometer that is no longer available for this research. In addition to filling this gap in available instrument time, the proposed new spectrometer, a Bruker model ESP 300, will increase the sensitivity and provide operational modes not available to the older instrument. In the conventional EPR mode, experiments provide sensitivity to nanosecond rotational motions that characterize the conformational dynamics of small biological molecules (e.g., lipids) or of small domains within macromolecules. In the saturation transfer EPR mode, microsecond rotational motions are detected, thus characterizing the larger scale motions of macromolecules. Pulsed micro-wave and pulsed laser accessories will provide the capabilities to perform time-resolved EPR experiments in the microsecond and millisecond time scales, permitting the application of new techniques developed by some of the applications. The principal biochemical and biophysical problems to be investigated will be: (1) the orientation and rotational motions of myosin and actin that are involved in the molecular mechanism of muscle contraction, (2) the role of lipid and protein dynamics in the mechanism of active calcium transport in sarcoplasmic reticulum membranes, (3) the role of lipid dynamics in malignant hyperthermia, (4) the conformational dynamics of alpha-2-macroglobulin, (5) the role of thioredoxin reductase in the reduction of free radicals in skin, and (6) the effects of steroids on membrane fluidity in granulocytes.
| Lewis, S M; Thomas, D D (1991) Microsecond rotational dynamics of spin-labeled Ca-ATPase during enzymatic cycling initiated by photolysis of caged ATP. Biochemistry 30:8331-9 |
| Berger, C L; Thomas, D D (1991) Rotational dynamics of actin-bound intermediates in the myosin ATPase cycle. Biochemistry 30:11036-45 |
| Fajer, P G; Fajer, E A; Thomas, D D (1990) Myosin heads have a broad orientational distribution during isometric muscle contraction: time-resolved EPR studies using caged ATP. Proc Natl Acad Sci U S A 87:5538-42 |
| Barnett, V A; Thomas, D D (1989) Microsecond rotational motion of spin-labeled myosin heads during isometric muscle contraction. Saturation transfer electron paramagnetic resonance. Biophys J 56:517-23 |
