Site-specific mutations in the myosin binding sites of actin affect structural transitions that control myosin binding.
We have examined the effects of actin mutations on myosin binding, detected by cosedimentation, and actin structural dynamics, detected by spectroscopic probes. Specific mutations were chosen that have been shown to affect the functional interactions of actin and myosin, two mutations (4Ac and E99A/E100A) in the proposed region of weak binding to myosin and one mutation (I341A) in the proposed region of strong binding. In the absence of nucleotide and salt, S1 bound to both wild-type and mutant actins with high affinity (K(d) < microM), but either ADP or increased ionic strength decreased this affinity. This decrease was more pronounced for actins with mutations that inhibit functional interaction with myosin (E99A/E100A and I341A) than for a mutation that enhances the interaction (4Ac). The mutations E99A/E100A and I341A affected the microsecond time scale dynamics of actin in the absence of myosin, but the 4Ac mutation did not have any effect. The binding of myosin eliminated these effects of mutations on structural dynamics; i.e., the spectroscopic signals from mutant actins bound to S1 were the same as those from wild-type actin. These results indicate that mutations in the myosin binding sites affect structural transitions within actin that control strong myosin binding, without affecting the structural dynamics of the strongly bound actomyosin complex.
Prochniewicz
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Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis 55455, USA. ewa@ddt.biochem.umn.edu
Thomas
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D000199:Actins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D000244:Adenosine Diphosphate / Q000737:chemistry; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D005454:Fluorescence Polarization; D008163:Luminescent Measurements; D046911:Macromolecular Substances; D020409:Molecular Motor Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018482:Muscle, Skeletal / Q000737:chemistry; D016297:Mutagenesis, Site-Directed; D015879:Myosin Subfragments / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics; D011817:Rabbits; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000235:genetics; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics
D000199:Actins; D046911:Macromolecular Substances; D020409:Molecular Motor Proteins; D015879:Myosin Subfragments; D000244:Adenosine Diphosphate