Myosin S1 changes the orientation of caldesmon on actin.
Changes in the orientation of caldesmon bound to actin in skeletal ghost myofibrils caused by the binding of myosin subfragment 1 (S1) were measured by fluorescence-detected linear dichroism using fluorescence microscopy. Gizzard caldesmon, labeled with acrylodan at its two Cys residues (CaD*), bound to actin with a probe angle that was unaffected by actin-bound tropomyosin. Irrigation of fibrils with myosin S1 dissociated most of the bound CaD*, but reintroduction of CaD* resulted in its rebinding to actin, without dissociation of S1, with a 7 degrees difference in probe angle. A similar change in probe angle was also observed when a 27-kDa actin-binding C-terminal fragment of caldesmon, labeled with acrylodan at its single Cys 580 (CaD-27*), was used. Introducing MgADP, which bound to S1 in the CaD*-actin-S1 ternary complex in the fibril, reversed the bound CaD* dichroism. These results indicate that (i) myosin heads and caldesmon compete for a common actin binding site; (ii) a ternary complex of CaD*-actin-S1 can be formed with an orientation of CaD* different from that in the CaD*-actin binary complex, and (iii) MgADP, which binds to and reorients myosin S1, affects the orientation of CaD* in the ternary complex. These results are consistent with a two-state binding model of caldesmon for actin in which state 1 involves a site that is competitive with S1 binding and state 2 involves a site that is formed in the presence of bound S1.
Szczesna
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Boston Biomedical Research Institute, Massachusetts 02114.
Graceffa
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Wang
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CL
Lehrer
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D000199:Actins / Q000737:chemistry*; D000818:Animals; D001667:Binding, Competitive; D002148:Calmodulin-Binding Proteins / Q000737:chemistry*; D002645:Chickens; D002942:Circular Dichroism; D015879:Myosin Subfragments / Q000737:chemistry*; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence
D000199:Actins; D002148:Calmodulin-Binding Proteins; D015879:Myosin Subfragments