Engineering Approaches to Biomolecular Motors

Engineering Approaches to Biomolecular Motors: From in vitro to in vivo Poster Abstracts

21-POS Board 21 Modifying Actomyosin Function by Small-molecular Inhibitor Blebbistatin Mohammad A. Rahman , Alf Månsson. Linnaeus University, Kalmar, Småland, Sweden. Addition of small molecular substances could substitute genetic engineering for temporarily changing myosin motor function. One interesting compound is blebbistatin that inhibits myosin II motor activity. In muscle cells, the effect of actin-myosin sliding velocity is modulated by myosin regulatory light chain (RLC) phosphorylation1. Otherwise, evidence exists that blebbistatin stabilizes a start of the power-stroke, strongly attached, actomyosin state2. Here, in vitro motility assay studies are performed to investigate (a) whether the drug effect varies with myosin RLC phosphorylation in absence of the myofilament lattice of muscle and (b) if the effect on velocity is consistent with stabilization of the mentioned actomyosin state. Heavy meromyosin (HMM) from Rabbit skeletal muscle, with either phosphorylated or un- phosphorylated RLCs, was adsorbed to silanized surfaces for in vitro motility assays. We found that blebbistatin (1µM) inhibits sliding velocity to similar degree (~50 %) independent of phosphorylation status. The inhibition induced by blebbistatin (2µM) was doubled for an increase in ionic strength (60 to 130 mM) whereas the effect on velocity was similar at different MgATP concentrations (0.1 – 1 mM). The findings suggest that the blebbistatin effect on velocity does not depend on myosin RLC phosphorylation, consistent with the idea that such dependence in vivo is fully attributed to myosin head – backbone interactions (absent in vitro). Furthermore, modelling suggests that blebbistatin increases the population of a start-of-power stroke actomyosin state with possible physiological roles e.g. in eccentric contractions (stretch of active muscle). 1.Stewart, M., Franks-Skiba, K. & Cooke, R. (2009) J. Muscle Res. Cell Motil. 30, 17–27.2.Takács, B. et al. (2010). Proc. Natl. Acad. Sci. 107, 6799–6804 Supported by the FET-program of EU-FP7 (grant agreement 613044; ABACUS)

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