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

Marko Ušaj,Venukumar Vemula,Alf Månsson,Aseem Salhotra,Luisa Moretto

doi:10.1038/s42003-020-01574-0

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.

Highlights

Benefits of single molecule studies of biomolecules include the need for minimal amounts of material and the potential to reveal phenomena hidden in ensembles
The first fluorescence-based single biomolecule assay[1] used total internal reflection fluorescence (TIRF) microscopy to study the turnover of a fluorescent ATP analog by the mechano-enzyme myosin II
Recent work[7] associated the fast processes with myosin conformers having different catalytic activity. Both for the general usefulness of single-molecule data in applications, e.g., drug screening and for deriving reliable mechanistic information, it is essential with unequivocal understanding of the fast processes in relation to enzymatic events with kcat of 0.05–0.1 s−1 according to solution kinetics[10,11]

Summary

Results and discussion

Fast rate processes in single-molecule studies under in vitro motility assay conditions. We hypothesized that UFOs on the motor adsorbing surfaces (Supplementary Fig. S2) was one of the artifacts contributing to the dominance of fast exponential processes in the analysis above Because such UFOs are stationary, they could be mistaken for hotspots attributed to myosin motor domains. The potential kinetic effect is evident from a simplified theoretical treatment, assuming monoexponential bleaching and blinking processes Under such conditions, the decay rate constant for the observed Alexa-ATP on times on myosin would be the sum of kcat, and the rate constants for photobleaching and photoblinking. If photoblinking and/or photobleaching are important one would, a priori, expect a reduction in rate and amplitude of the fast processes[24] with reduced intensity, as well as possible reemergence of the slow process corresponding to catalytic events when performing the assay in IVMA solution Such effects were not clearly observed (Supplementary Fig. S3a, b).

IVMA buffer TIRFM buffer

5.24 TIRF buffer b

A ATPase2 e