Studies of the interaction between titin and myosin.
The interaction of titin with myosin has been studied by binding assays and electron microscopy. Electron micrographs of the titin-myosin complex suggest a binding site near the tip of the tail of the myosin molecule. The distance from the myosin head-tail junction to titin indicates binding 20-30 nm from the myosin COOH terminus. Consistent with this, micrographs of titin-light meromyosin (LMM) show binding near the end of the LMM molecule. Plots of myosin- and LMM-attachment positions along the titin molecule show binding predominantly in the region located in the A band in situ, which is consistent with the proposal that titin regulates thick filament assembly. Estimates of the apparent dissociation constant of the titin-LMM complex were approximately 20 nM. Assays of LMM cyanogen bromide fragments also suggested a strong binding site near the COOH terminus. Proteolysis of a COOH-terminal 17.6-kD CNBr fragment isolated from whole myosin resulted in eight peptides of which only one, comprising 17 residues, bound strongly to titin. Two isoforms of this peptide were detected by protein sequencing. Similar binding data were obtained using synthetic versions of both isoforms. The peptide is located immediately COOH-terminal of the fourth "skip" residue in the myosin tail, which is consistent with the electron microscopy. Skip-4 may have a role in determining thick filament structure, by allowing abrupt bending of the myosin tail close to the titin-binding site.
Department of Veterinary Clinical Sciences, Bristol University, Langford, United Kingdom.
A
A
Houmeida
J
J
Holt
L
L
Tskhovrebova
J
J
Trinick
D000595:Amino Acid Sequence; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D064211:Connectin; D003488:Cyanogen Bromide; D004347:Drug Interactions; D007144:Immunoglobulin J-Chains / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism* / Q000648:ultrastructure; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000378:metabolism* / Q000648:ultrastructure; D015879:Myosin Subfragments / Q000378:metabolism / Q000648:ultrastructure; D009218:Myosins / Q000378:metabolism* / Q000648:ultrastructure; D011485:Protein Binding / Q000502:physiology; D011494:Protein Kinases / Q000378:metabolism* / Q000648:ultrastructure; D011817:Rabbits
D064211:Connectin; D007144:Immunoglobulin J-Chains; D008565:Membrane Proteins; D009124:Muscle Proteins; D015879:Myosin Subfragments; D011494:Protein Kinases; D009218:Myosins; D003488:Cyanogen Bromide