Blebbistatin Effects Expose Hidden Secrets in the Force-Generating Cycle of Actin and Myosin

Abstract

Cyclic interactions between myosin II motors and actin filaments driven by ATP turnover underlie muscle contraction and have key roles in the motility of nonmuscle cells. A remaining enigma in the understanding of this interaction is the relationship between the force-generating structural change and the release of the ATP-hydrolysis product, inorganic phosphate (Pi), from the active site of myosin. Here, we use the small molecular compound blebbistatin to probe otherwise hidden states and transitions in this process. Different hypotheses for the Pi release mechanism are tested by interpreting experimental results from in vitro motility assays and isolated muscle fibers in terms of mechanokinetic actomyosin models. The data fit with ideas that actomyosin force generation is preceded by Pi release, which in turn is preceded by two serial transitions after/coincident with cross-bridge attachment. Blebbistatin changes the rate limitation of the cycle from the first to the second of these transitions, uncovering functional roles of an otherwise short-lived pre-power stroke state that has been implicated by structural data.

The effects of inorganic phosphate on muscle force development and energetics: challenges in modelling related to experimental uncertainties

“…It is shown above that models assuming unbranched pathways, tight coupling between force-generation and biochemical transitions and no slippage of cross-bridges between sites, account quite well for a range of experimental data at varied [Pi]. Particularly successful in this regard is the 10:1 model similar to that of Rahman et al (2018), found to account for a range of blebbistatin effects in addition to data obtained under physiological conditions. A challenge for all models tested is the anomalous force-velocity relationship predicted to appear at high loads (low velocity) when [Pi] is increased.…”

“…ATP turnover rate and power output) during shortening under different conditions. Additionally, effects of the small molecular substance blebbistatin are well predicted by the 10:1 model (Rahman et al 2018). The recent modelling studies have focused on steady-state experiments.…”

“…Major strengths of all versions of the models tested here are independent origin of model parameter values primarily from biochemical and single molecule studies of actin and myosin, as described by Månsson (2016) and Rahman et al (2018). Parameter values have further been obtained under Potma et al 1995).…”

“…Furthermore, arguments have also been put forward (Månsson 2019) to suggest that the tension transient in response to fast length steps, with the T1 and T2 relationships (Huxley and Simmons 1971), should be consistent with the model. Eccentric contractions however, seem to involve complexities that require additional assumptions in the models which do not follow directly from experimental findings of isolated proteins (Campbell et al 2011;Rahman et al 2018;Rassier and Pavlov 2012). One may argue that the number of states is too high in the "simple" models to denote these models as "simple".…”

Optimized single molecule fluorescence sheds light on elusive enzymatic mechanisms

“…Myosin, heavy meromyosin (HMM) and papain subfragment 1 (S1) were prepared following published protocols ((48), with modifications in (46,49)). Protein preparations were characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) with respect to purity as described previously (50) and concentrations were determined spectrophotometrically. TIRF Microscopy.…”