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Visualization of domains in native and nucleotide-trapped myosin heads by negative staining
1Muscle Biology Department, AFRC Institute of Food Research, Bristol Laboratory, Langford.
Abstract:
Electron microscopy of negatively stained vertebrate skeletal muscle myosin molecules has revealed substructure suggestive of globular domains in the head portions of the molecule. This head substructure has been examined after both low and high electron doe. The results suggest it is probably not an artefact of radiation damage. The most common appearance is of one or two stain-filled clefts which run roughly perpendicular to the long axis of the head, giving rise to the appearance of two or three domains in a line. A large domain is located at the end of the head, while two smaller domains are arranged between this and the head-tail junction. The size of the large distal domain (about 10 nm long and about 7 nm wide at its widest point) is similar in heads showing either two or three domains. Stable analogues of M.ATP and M.ADP.Pi, the predominant complexes present during hydrolysis of ATP by myosin, were prepared by crosslinking the two reactive SH groups (SH1 and SH2) in the myosin head heavy chain with N,N'-p-phenylenedimaleimide (pPDM) in the presence of ADP, and by forming a complex with vanadate ion and ADP. At this resolution (approximately 2 nm) the heads of these modified molecules did not appear markedly different from those of the untreated protein, although there was a small increase in the number of straight as opposed to curved heads after cross-linking with pPDM.
Insights
Electron microscopy reveals substructure in vertebrate skeletal muscle myosin heads, showing globular domains. Stable myosin analogues prepared via cross-linking or vanadate complexation showed minimal structural changes.
Area of Science:
- Biochemistry
- Molecular Biology
- Structural Biology
Background:
- Vertebrate skeletal muscle myosin is crucial for muscle contraction.
- Understanding myosin head substructure provides insights into its mechanical function.
- Previous studies suggested globular domains within the myosin head.
Purpose of the Study:
- To investigate the substructure of vertebrate skeletal muscle myosin heads using electron microscopy.
- To determine if observed substructure is an artifact of radiation damage.
- To examine structural changes in myosin heads upon formation of stable nucleotide analogues.
Main Methods:
- Negatively stained electron microscopy of myosin molecules.
- Examination of myosin head substructure at low and high electron doses.
- Preparation of stable myosin analogues using N,N'-p-phenylenedimaleimide (pPDM) cross-linking of SH groups in the presence of ADP.
- Formation of myosin-ADP-vanadate complexes.
Main Results:
- Electron microscopy revealed substructure in myosin heads, consistent with globular domains.
- The observed substructure was unlikely to be an artifact of radiation damage.
- Myosin heads commonly displayed one or two stain-filled clefts, appearing as two or three domains.
- A large distal domain (approx. 10 nm x 7 nm) was identified.
- Stable analogues of M.ATP and M.ADP.Pi showed minimal structural differences from untreated myosin at ~2 nm resolution.
- pPDM cross-linking resulted in a slight increase in straight myosin heads.
Conclusions:
- Vertebrate skeletal muscle myosin heads possess a distinct substructure composed of globular domains.
- This substructure is likely a genuine feature of the myosin molecule, not a radiation artifact.
- Formation of stable nucleotide analogues does not significantly alter the overall domain structure of the myosin head at this resolution.