In Vitro Motility Assay Studies at Low [MgATP] - Evidence For Inter-Head Cooperativity in Fast Skeletal Myosin II

• The idea that fast skeletal myosin II exhibits processivity with sequential actions of the two myosin heads during muscle contraction has long been a topic of discussion but with appreciable difficulties to obtain conclusive experimental results. The difficulties may be related to a limited processivity (few sequential head actions) that is of greatest importance at low velocity (e.g. high resistive load), difficult to study accurately in vitro. In order to aid the investigations we here make use of recent evidence that the bipyridine drug amrinone inhibits the strain-dependent ADP-release step of myosin II with expected enhancement of processivity (Albet-Torres et al., J Biol Chem., 2009); Månsson, Biophys J., 2010). For accurate velocity measurements, the recombinant expressed capping protein CapZ (Soeno et. al., J Muscle Res Cell Motil., 1998) was fluorescence labeled. Nanometer tracking was achieved by two-dimensional Gaussian fits to single molecule fluorescence intensity profiles representing CapZ attached to the trailing actin filament end. Importantly, the heavy meromyosin (HMM) propelled actin filament sliding velocity (1mM MgATP) for CapZ-capped filaments was comparable to that of uncapped filaments at different ionic strengths and HMM surface densities. Studies at low [MgATP] (5-30mM) suggests a non-linearity in the [MgATP]-velocity plot that was enhanced by 1mM amrinone. The result is in contrast to the linearity expected for independent myosin heads and could be interpreted as an apparent velocity dependence of the myosin step length. This is in accordance with the idea of limited processivity of myosin II if it is assumed that a doubled apparent step length corresponds to sequential action of the two heads. Further insight into the mechanism will be obtained using proteolytically prepared one-headed HMM and e.g. studies with external loads on actin.

Ämnesord

NATURVETENSKAP  -- Biologi -- Biofysik (hsv//swe)

NATURAL SCIENCES  -- Biological Sciences -- Biophysics (hsv//eng)

NATURVETENSKAP  -- Biologi -- Biokemi och molekylärbiologi (hsv//swe)

NATURAL SCIENCES  -- Biological Sciences -- Biochemistry and Molecular Biology (hsv//eng)

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