Myosin motors with artificial lever arms.
The myosin head consists of a globular catalytic domain and a light chain binding domain (LCBD). The coupling efficiency between ATP hydrolysis and myosin-induced actin movement is known to decline as the LCBD is truncated or destabilized. However, it was not clear whether the observed alteration in the production of force and movement reflects only the mechanical changes to the length of the LCBD or whether these changes also affect the kinetic properties of the catalytic domain. Here we show that replacement of the LCBD with genetically engineered domains of similar rigidity and dimensions produces functional molecular motors with unchanged kinetic properties. The resulting single-chain, single-headed motors were produced in Dictyostelium discoideum and obtained after purification from a standard peptone-based growth medium at levels of up to 12 mg/l. Their actin motility properties are similar or greater than those of native myosin. Rates of 2.5 and 3.3 microm/s were observed for motor domains fused to one or two of these domains, respectively. Their kinetic and functional similarity to the extensively studied myosin subfragment 1 (S1) and their accessibility to molecular genetic approaches makes these simple constructs ideal models for the investigation of chemo-mechanical coupling in the myosin motor.
Anson
M
M
National Institute for Medical Research, London, UK.
Geeves
M A
MA
Kurzawa
S E
SE
Manstein
D J
DJ
D008841:Actin Cytoskeleton / Q000378:metabolism; D000185:Actinin / Q000235:genetics / Q000378:metabolism; D000199:Actins / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D004023:Dictyostelium / Q000737:chemistry*; D007700:Kinetics; D013058:Mass Spectrometry; D008958:Models, Molecular; D018994:Myosin Light Chains / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D011485:Protein Binding; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence
D000199:Actins; D018994:Myosin Light Chains; D011994:Recombinant Proteins; D000185:Actinin; D000255:Adenosine Triphosphate