The orientation of spin-probes attached to Cys374 on actin in oriented gels

Changes in protein orientation are thought to be involved in the generation of force that occurs in active muscle. To address the possibility that such changes occur in the actin filament, a spin label was attached to Cys374 of actin and its orientation was measured for filaments that were aligned in capillaries. In the best aligned samples the label was found in two populations, one with a narrow distribution of angles centered at an average angle of 36 (+/- 4) degrees, and a second in a broad distribution centered at an average angle of 45(+/- 4) degrees. The birefringence of the samples was high and X-ray diffraction patterns showed that the actin filaments were well aligned with average angles similar to the population of the better aligned spin labels, suggesting that the more disordered population of probes arose from either disorder of the probe relative to the protein, or from disorder of a local portion of the protein. When myosin subfragment-1 (S-1) was also included with the actin, molar ratio 1 S-1 to 4 actins, there was little change in the angular distribution of either of the two probe populations. We conclude that the binding of the myosin head does not produce appreciable alterations in the orientation of this domain.