Differential dynamic behavior of actin filaments containing tightly-bound Ca2+ or Mg2+ in the presence of myosin heads actively hydrolyzing ATP.
We have investigated how Ca2+ or Mg2+ bound at the high-affinity cation binding site in F-actin modulates the dynamic response of these filaments to ATP hydrolysis by attached myosin head fragments (S1). Rotational motions of the filaments were monitored using steady-state phosphorescence emission anisotropy of the triplet probe erythrosin-5-iodoacetamide covalently attached to cysteine 374 of actin. The anisotropy of filaments containing only Ca2+ increased from 0.080 to 0.137 upon binding S1 in a rigor complex and decreased to 0.065 in the presence of ATP, indicating that S1 induced additional rotational motions in the filament during ATP hydrolysis. The comparable anisotropy values for Mg(2+)-containing filaments were 0.067, 0.137, and 0.065, indicating that S1 hydrolysis did not induce measurable rotational motions in these filaments. Phalloidin, a fungal toxin which stabilizes F-actin and increases its rigidity, increased the anisotropy of F-actin containing either Ca2+ or Mg2+ but not the anisotropy of the 1:1 S1-actin complexes of these filaments. Mg(2+)-containing filaments with phalloidin bound also displayed increased rotational motions during S1 ATP hydrolysis. A strong positive correlation between the phosphorescence anisotropy of F-actin under specific conditions and the extent of the rotational motions induced by S1 during ATP hydrolysis suggested that the long axis torsional rigidity of F-actin plays a crucial role in modulating the dynamic response of the filaments to ATP hydrolysis by S1. Cooperative responses of F-actin to dynamic perturbations induced by S1 during ATP hydrolysis may thus be physically mediated by the torsional rigidity of the filament.
Rebello
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CA
Department of Food Science, Rutgers, The State University, New Brunswick, New Jersey 08901-8520, USA.
Ludescher
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RD
D008841:Actin Cytoskeleton / Q000737:chemistry / Q000378:metabolism; D000199:Actins / Q000737:chemistry* / Q000378:metabolism; D000205:Actomyosin / Q000737:chemistry / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D005260:Female; D005454:Fluorescence Polarization; D006868:Hydrolysis; D008163:Luminescent Measurements; D046911:Macromolecular Substances; D008274:Magnesium / Q000378:metabolism*; D008297:Male; D020409:Molecular Motor Proteins / Q000737:chemistry* / Q000378:metabolism; D015879:Myosin Subfragments / Q000737:chemistry / Q000378:metabolism*; D009994:Osmolar Concentration; D011485:Protein Binding; D011817:Rabbits
D000199:Actins; D046911:Macromolecular Substances; D020409:Molecular Motor Proteins; D015879:Myosin Subfragments; D000255:Adenosine Triphosphate; D000205:Actomyosin; D008274:Magnesium; D002118:Calcium