Moretto, L. | Search bioRxiv

bioRxiv2023

New paradigms in actomyosin energy transduction: critical evaluation of non-traditional models for orthophosphate release

Release of the ATP hydrolysis product inorganic phosphate (Pi) from the active site of myosin is central in chemo-mechanical energy transduction and closely associated with the main force-generating structural change, the power-stroke. Despite intense investigations, the relative timing between Pi-release and the power-stroke remains poorly understood. This hampers in depth understanding of the production of force and motion by myosin in health and disease and also our understanding of myosin-active drugs. From the 1990s and up to today, models with the Pi-release either distinctly before or after the power-stroke, in unbranched kinetic schemes, have dominated the literature. However, in recent years, alternative models have emerged to explain apparently contradictory findings. Here, we first compare and critically analyze, three influential alternative models, either characterized by a branched kinetic scheme or by partial uncoupling of Pi-release and the power-stroke. Finally, we suggest critical tests of the models aiming for a unified picture.

biophysics↗

bioRxiv2020

Optimized single molecule fluorescence sheds light on elusive enzymatic mechanisms

Single molecule enzymology using fluorescent substrate requires truly minimal amounts of proteins. This is highly beneficial when the protein source is either advanced expression systems or samples from humans/animals with ethical and economic implications. Further benefits of single molecule analysis is the potential to reveal phenomena hidden in ensemble studies. However, dye photophysics and fluorescent contaminants complicate interpretation of the single molecule data. We here corroborate the importance of such complexities using fluorescent Alexa647 ATP to study ATP turnover by myosin and actomyosin. We further show that the complexities are largely eliminated by aggressive surface cleaning and use of a range of triple state quenchers and redox agents with minor effects on actin-myosin function. Using optimized assay conditions, we then show that the distributions of ATP binding dwell times on myosin are best described by the sum of 2 to 3 exponential processes. This applies in the presence and absence of actin and in the presence and absence of the drug para-aminoblebbistatin. Two of the processes are attributable to ATP turnover by myosin and actomyosin, respectively. A remaining process with rate constant in the range 0.2-0.5 s-1 is consistent with non-specific ATP binding to myosin and bioinformatics modelling suggests that such binding may be important for accelerated ATP transport to the active site. Finally, we report studies of the actin-activated myosin ATP turnover under conditions with no sliding between actin and myosin, as in isometrically contracting muscle, revealing heterogeneity in the ATP turnover kinetics between different molecules.

biophysics↗