Single molecule turnover of fluorescent ATP by myosin and actomyosin unveil elusive enzymatic mechanisms

Abstract

Benefits of single molecule studies of biomolecules include the need for minimal amounts of material and the potential to reveal phenomena hidden in ensembles. However, results from recent single molecule studies of fluorescent ATP turnover by myosin are difficult to reconcile with ensemble studies. We found that key reasons are complexities due to dye photophysics and fluorescent contaminants. After eliminating these, through surface cleaning and use of triple state quenchers and redox agents, the distributions of ATP binding dwell times on myosin are best described by 2 to 3 exponential processes, with and without actin, and with and without the inhibitor para-aminoblebbistatin. Two processes are attributable to ATP turnover by myosin and actomyosin respectively, whereas the remaining process (rate constant 0.2–0.5 s−1) is consistent with non-specific ATP binding to myosin, possibly accelerating ATP transport to the active site. Finally, our study of actin-activated myosin ATP turnover without sliding between actin and myosin reveals heterogeneity in the ATP turnover kinetics consistent with models of isometric contraction.

Multistep orthophosphate release tunes actomyosin energy transduction

“…3 ) of the myosin head at the exit of the back door suggests the possibility of additional electrostatic Pi-binding. This is in accordance with what we hypothesized above based on recent evidence 35 suggesting nonspecific binding of fluorescent ATP to the myosin motor domain. The latter type of Pi-binding should, if existing, be readily accessible to experimental verification in contrast to binding to the secondary site considered above.…”

“…Another possibility is additional Pi-binding sites outside the Pi-release tunnel, on the surface of the myosin motor domain. This idea is consistent with recent observations 35 suggesting nonspecific binding of fluorescent ATP to myosin motor domains, effects that may partly be mediated via the phosphate groups of ATP. However, to the best of our knowledge, such Pi-binding sites have not been explicitly demonstrated, and, if they exist, their contribution to a delayed Pi-appearance in solution is unclear.…”

“…2c, d ). Recent experimental results, comparing isolated HMM and myosin subfragment 1 motor domains, convincingly attributed the fast phases to ATP binding to the myosin head outside the active site 35 . It is of interest to note that similar auxiliary ATP binding sites have recently been demonstrated in another motor protein 39 .…”

Critical Evaluation of Current Hypotheses for the Pathogenesis of Hypertrophic Cardiomyopathy

“…Additionally, optical tweezers and cantilever-based force measurements can be used to directly measure the forces developed by single myosin motors or small motor ensembles when they interact with an actin filament or a thin filament (actin + regulatory proteins) [99,112]. Moreover, myosin and actomyosin ATP turnover kinetics can be probed by single-molecule ATPase assays [113,114], using total internal reflection fluorescence (TIRF) microscopy and fluorescent ATP. Proteins, both with and without mutations, can be isolated from hearts of patients with HCM and donor hearts, respectively.…”

“…The idea of increased cross-bridge detachment rate with several HCM mutations is also consistent with increased rate of relaxation found in myofibrils affected by different HCM mutations after isometric contractions at full calcium-activation (see References [81,130,195]; reviewed in References [21,48]). Interestingly, this effect would also increase the ATP turnover rate during isometric contraction and other contractions at high tension levels where the ATP turnover rate is limited by the cross-bridge detachment rate [21,114]. These tension levels are those where the heart operates during a large fraction of its working cycle.…”