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Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae
Published on: July 9, 2013
Myosin II folding is mediated by a molecular chaperonin
1Department of Pathology and Laboratory Medicine, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, Piscataway, New Jersey 08854, USA.
The Journal of Biological Chemistry
|September 10, 1999
Summary
Myosin motor protein folding is aided by chaperonins and muscle-specific factors. The myosin motor domain
Area of Science:
- Biochemistry
- Molecular Biology
- Muscle Physiology
Background:
- Skeletal muscle myosin's heavy meromyosin subfragment (HMM) is crucial for muscle contraction.
- Understanding myosin folding is key to muscle function and disease research.
Purpose of the Study:
- To investigate the in vitro folding pathway of skeletal muscle myosin's HMM.
- To identify key protein factors and intermediates involved in myosin assembly.
Main Methods:
- In vitro synthesis of myosin heavy and light chains using coupled transcription-translation.
- Analysis of nascent translation products for folding intermediates.
- Biochemical and functional assays to assess folding completeness.
- Preparation and use of cytoplasmic extract from mouse myogenic cell line.
Main Results:
- A major folding intermediate was identified, complexed with eukaryotic cytosolic chaperonin.
- HMM release from the chaperonin complex is ATP-dependent but results in incomplete motor domain folding.
- Myosin heavy chain dimerization and heavy-light chain association occur early.
- A cytoplasmic extract significantly enhanced HMM folding, indicating muscle-specific factors are involved.
Conclusions:
- Eukaryotic cytosolic chaperonin mediates myosin molecular assembly.
- Myosin motor domain folding is the rate-limiting step in the pathway.
- Muscle-specific factors play a significant role in complete myosin folding.
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