Conformational Studies of Myosin and Actin with Calibrated Resonance Energy Transfer

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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 of versions of the swinging lever arm model, and indicate that actin participation is indispensable for conformational changes leading to force generation. The conformational selection during weak binding at the actomyosin interface may precock the myosin head for the ensuing powerstroke.

Creator(s): Xu, Jin
Creation Date: May 2000
Partner(s):
UNT Libraries
Collection(s):
UNT Theses and Dissertations
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Total Uses: 67
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Publisher Info:
Publisher Name: University of North Texas
Place of Publication: Denton, Texas
Date(s):
  • Creation: May 2000
  • Digitized: June 28, 2007
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 of versions of the swinging lever arm model, and indicate that actin participation is indispensable for conformational changes leading to force generation. The conformational selection during weak binding at the actomyosin interface may precock the myosin head for the ensuing powerstroke.

Degree:
Level: Doctoral
Discipline: Biochemistry
Language(s):
Subject(s):
Keyword(s): Actomyosin | ATP hydrolysis | Resonance energy transfer
Contributor(s):
Partner:
UNT Libraries
Collection:
UNT Theses and Dissertations
Identifier:
  • OCLC: 47266047 |
  • UNTCAT: b2302729 |
  • ARK: ark:/67531/metadc2438
Resource Type: Thesis or Dissertation
Format: Text
Rights:
Access: Use restricted to UNT Community
License: Copyright
Holder: Xu, Jin
Statement: Copyright is held by the author, unless otherwise noted. All rights reserved.