Description: Resonance energy transfer was employed to study the conformational changes of actomyosin during ATP hydrolysis. To calibrate the technique, the parameters for resonance energy transfer were defined. With conformational searching algorithms to predict probe orientation, the distances measured by resonance energy transfer are highly consistent with the atomic models, which verified the accuracy and feasibility of resonance energy transfer for structural studies of proteins and oligonucleotides. To study intramyosin distances, resonance energy transfer probes were attached to skeletal myosin's nucleotide site, subfragment-2, and regulatory light chain to examine nucleotide analog-induced structural transitions. The distances between the three positions were measured in the presence of different nucleotide analogs. No distance change was considered to be statistically significant. The measured distance between the regulatory light chain and nucleotide site was consistent with either the atomic model of skeletal myosin subfragment-1 or an average of the three models claimed for different ATP hydrolysis states, which suggested that the neck region was flexible in solution. To examine the participation of actin in the powerstroke process, resonance energy transfer between different sites on actin and myosin was measured in the presence of nucleotide analogs. The efficiencies of energy transfer between myosin catalytic domain and actin were consistent with the actoS1 docking model. However, the neck region was much closer to the actin filament than predicted by static atomic models. The efficiency of energy transfer between Cys 374 and the regulatory light chain was much greater in the presence of ADP-AlF4, ADP-BeFx, and ADP-vanadate than in the presence of ADP or no nucleotide. These data detect profound differences in the conformations of the weakly and strongly attached crossbridges which appear to result from a conformational selection that occurs during the weak binding of the myosin head to actin. The resonance energy transfer data exclude a number ...
Date: May 2000
Creator: Xu, Jin
Item Type: Thesis or Dissertation
Partner: UNT Libraries