A 29 residue region of the sarcomeric myosin rod is necessary for filament formation.
Myosin is a motor protein whose functional unit in the sarcomere is the thick filament. The myosin molecule is capable of self-assembly into thick filaments through its alpha-helical coiled-coil rod domain. To define more precisely the sequence requirements for this assembly, segments of the human fast IId skeletal myosin rod were expressed in Escherichia coli and examined differential solubility and the formation of ordered paracrystals. We show that both properties appear to require a 29 residue sequence (residues 1874 to 1902) near the C terminus of the rod region. To test further the role of this region in assembly, a protein was constructed which consisted of this assembly competence domain (ACD) fused to the carboxy terminus of an assembly-incompetent myosin rod fragment. This chimeric fragment exhibited myosin's characteristic solubility properties and formed ordered paracrystals. To complement these in vitro experiments, both a full-length myosin heavy chain (MYH) and one from which the 29 residues were deleted were transfected into cultured mammalian cells. While the full-length construct formed the spindle-shaped structures characteristic of arrays of thick filaments, the deleted MYH showed only diffuse staining throughout the cytoplasm by light microscopy. Thus, there appears to be a specific sequence in the C-terminal region of the myosin heavy chain rod which is necessary for ordered paracrystal formation and is sufficient to confer assembly properties to an assembly-incompetent rod fragment.
Sohn
R L
RL
Department of Molecular, Cellular and Developmental Biology, Universityof Colorado, Boulder 80309-0347, USA.
Vikstrom
K L
KL
Strauss
M
M
Cohen
C
C
Szent-Gyorgyi
A G
AG
Leinwand
L A
LA
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D019556:COS Cells / Q000378:metabolism; D003460:Crystallization; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008854:Microscopy, Electron; D008958:Models, Molecular; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000648:ultrastructure; D018995:Myosin Heavy Chains / Q000737:chemistry / Q000235:genetics / Q000648:ultrastructure; D018994:Myosin Light Chains / Q000737:chemistry / Q000235:genetics / Q000648:ultrastructure; D009218:Myosins / Q000737:chemistry* / Q000235:genetics / Q000648:ultrastructure*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure; D017384:Sequence Deletion; D013329:Structure-Activity Relationship
D018994:Myosin Light Chains; D011994:Recombinant Proteins; D018995:Myosin Heavy Chains; D009218:Myosins