Optimized single molecule fluorescence sheds light on elusive enzymatic mechanisms

Abstract

Author ContributionsA.M. and M.U. conceived the project, M.U. and A.M. designed the TIRF system. M.U. and A.M. designed the experiments. M.U. built the TIRF system and performed single molecule fluorescence experiments. M.U. L.M. and A.M. analyzed TIRF data, L.M. performed the bioinformatics modelling. V.V. performed in vitro motility assay experiments with and without AmBleb and analyzed the data, and A.S. analyzed in vitro motility assay data, A.M., M.U., L.M., V.V. and A.S. wrote the manuscript and approved the final version. AbstractSingle 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.

Myosin and gelsolin cooperate in actin filament severing and actomyosin motor activity

“…TIRF assay TIRF assay solution, pH 7.4 was prepared to contain (final concentrations): 2 mM Trolox, 2 mM Cyclooctatetraene (COT), 2 mM 4-Nitrobenzyl alcohol (NBA), 10 mM DTT, 45 mM KCl, 7.2 mg/ml glucose, 3 U/ml pyranose oxidase (POX), 0.01 mg/ml catalase, 2.5 mM creatine phosphate and 0.2 mg/ml creatine phosphokinase in LISS [99]. Initially, 100 mM Trolox stock was prepared in methanol, followed by dilution in LISS, subsequent filtering through a 0.2 µm filter and exposure to UV light (254 nm, 120,000 µJ/cm 2 ) for 15 min to form Trolox-Quinone.…”

Myosin-gelsolin cooperativity in actin filament severing and actomyosin motor activity

“…TIRF assay TIRF assay solution, pH 7.4 was prepared to contain (final concentrations): 2 mM Trolox, 2 mM Cyclooctatetraene (COT), 2 mM 4-Nitrobenzyl alcohol (NBA), 10 mM DTT, 45 mM KCl, 7.2 mg/ml glucose, 3 U/ml POX, 0.01 mg/ml catalase, 2.5 mM creatine phosphate and 0.2 mg/ml creatine phosphokinase in LISS [61]. Initially, 100 mM Trolox stock was prepared in methanol, followed by dilution in LISS, subsequent filtering through a 0.2 µm filter and exposure to UV light (254 nm, 120,000 µJ/cm 2 ) for 15 min to form Trolox-Quinone.…”

“…The Trolox-Trolox/Trolox-Quinone mixture prepared in LISS (TT/TQ-LISS) was degassed before use. For further details, please see [61]. The TIRF assay was performed by first adsorbing HMM (12 µg/ml) onto TMCS-derivatized glass surfaces (5 min).…”

“…Subsequently, the flow-cell was infused sequentially with 1 mg/ml nonfluorescent BSA (2 min), wash buffer L65, rhodamine-phalloidin labelled F-actin (5 nM, 2 min) and gelsolin (5 nM, labelled with Alexa-647) with varying concentrations of free calcium (see above) as desired. All TIRF assay experiments were performed at a stable temperature (23 ± 1 °C), using an objective heater [61]. Time-lapse movies were acquired with an exposure time of 50 ms. An objective-based TIRF illumination system was built in house using a Nikon TIRF 60X objective (NA=1.49), a Nikon Eclipse TE300 inverted microscope, an Andor iXon Ultra 897 EMCCD camera and a 642 nm diode laser for illumination as described elsewhere [61].…”

“…All TIRF assay experiments were performed at a stable temperature (23 ± 1 °C), using an objective heater [61]. Time-lapse movies were acquired with an exposure time of 50 ms. An objective-based TIRF illumination system was built in house using a Nikon TIRF 60X objective (NA=1.49), a Nikon Eclipse TE300 inverted microscope, an Andor iXon Ultra 897 EMCCD camera and a 642 nm diode laser for illumination as described elsewhere [61]. The simultaneous addition of NBA, COT and Trolox, to improve dye photo-physical properties in the TIRF assay, reduced the actin filament sliding velocity whereas Trolox alone had negligible effects (compare Fig.…”