title: Independent movement of the regulatory and catalytic domains of myosin heads revealed by phosphorescence anisotropy.
abstract: Inter- and intradomain flexibility of the myosin head was measured using phosphorescence anisotropy of selectively labeled parts of the molecule. Whole myosin and the myosin head, subfragment-1 (S1), were labeled with eosin-5-iodoacetamide on the catalytic domain (Cys 707) and on two sites on the regulatory domain (Cys 177 on the essential light chain and Cys 154 on the regulatory light chain). Phosphorescence anisotropy was measured in soluble S1 and myosin, with and without F-actin, as well as in synthetic myosin filaments. The anisotropy of the former were too low to observe differences in the domain mobilities, including when bound to actin. However, this was not the case in the myosin filament. The final anisotropy of the probe on the catalytic domain was 0.051, which increased for probes bound to the essential and regulatory light chains to 0.085 and 0.089, respectively. These differences can be expressed in terms of a "wobble in a cone" model, suggesting various amplitudes. The catalytic domain was least restricted, with a 51 +/- 5 degrees half-cone angle, whereas the essential and regulatory light chain amplitude was less than 29 degrees. These data demonstrate the presence of a point of flexibility between the catalytic and regulatory domains. The presence of the "hinge" between the catalytic and regulatory domains, with a rigid regulatory domain, is consistent with both the "swinging lever arm" and "Brownian ratchet" models of force generation. However, in the former case there is a postulated requirement for the hinge to stiffen to transmit the generated torque associated by nucleotide hydrolysis and actin binding.
authors.affiliation: Department of Pathology, University of Sydney NSW 2006, Australia.
authors.forename: L J
authors.initials: LJ
authors.lastname: Brown
authors.forename: N
authors.initials: N
authors.lastname: Klonis
authors.forename: W H
authors.initials: WH
authors.lastname: Sawyer
authors.forename: P G
authors.initials: PG
authors.lastname: Fajer
authors.forename: B D
authors.initials: BD
authors.lastname: Hambly
mesh_terms: D000818:Animals; D020134:Catalytic Domain / Q000502:physiology*; D005454:Fluorescence Polarization / Q000379:methods; D008163:Luminescent Measurements; D020409:Molecular Motor Proteins / Q000737:chemistry* / Q000502:physiology*; D018482:Muscle, Skeletal / Q000737:chemistry / Q000502:physiology; D018994:Myosin Light Chains / Q000737:chemistry / Q000502:physiology; D015879:Myosin Subfragments / Q000737:chemistry* / Q000502:physiology*; D017434:Protein Structure, Tertiary; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship
chemical_list: D020409:Molecular Motor Proteins; D018994:Myosin Light Chains; D015879:Myosin Subfragments