Abstract
We have used a novel time-resolved FRET (TR-FRET) assay to detect small-molecule modulators of actin-myosin structure and function. Actin-myosin interactions play crucial roles in the generation of cellular force and movement. Numerous mutations and post-translational modifications of actin or myosin disrupt muscle function and cause life-threatening syndromes. Here, we used a FRET biosensor to identify modulators that bind to the actin-myosin interface and alter the structural dynamics of this complex. We attached a fluorescent donor to actin at Cys-374 and a nonfluorescent acceptor to a peptide containing the 12 N-terminal amino acids of the long isoform of skeletal muscle myosin's essential light chain. The binding site on actin of this acceptor-labeled peptide (ANT) overlaps with that of myosin, as indicated by (a) a similar distance observed in the actin-ANT complex as in the actin-myosin complex and (b) a significant decrease in actin-ANT FRET upon binding myosin. A high-throughput FRET screen of a small-molecule library (NCC, 727 compounds), using a unique fluorescence lifetime readout with unprecedented speed and precision, showed that FRET is significantly affected by 10 compounds in the micromolar range. Most of these "hits" alter actin-activated myosin ATPase and affect the microsecond dynamics of actin detected by transient phosphorescence anisotropy. We conclude that the actin-ANT TR-FRET assay enables detection of pharmacologically active compounds that affect actin structural dynamics and actomyosin function. This assay establishes feasibility for the discovery of allosteric modulators of the actin-myosin interaction, with the ultimate goal of developing therapies for muscle disorders.
Highlights
We have used a novel time-resolved FRET (TR-FRET) assay to detect small-molecule modulators of actin–myosin structure and function
Analysis of the FRET decay showed that the mean distance (R) between the two probes was 3.3 Ϯ 0.2 nm, in very good agreement with the distance detected for probes at Cys-374 on actin and on Cys-16 at the N-terminal extension (NTE) of A1 [4]
The addition of ATP to a nucleotide-free mixture of actin, ANT, and subfragment 1 (S1) increased FRET between actin and ANT for the NTE-containing S1 isoforms, but not for S1A2 (Fig. 3B). This result serves as further evidence for overlapping ANT and NTE binding sites, as ATP causes dissociation of all S1 constructs from actin
Summary
Time-resolved fluorescence decays of donor-labeled actin in the presence of increasing concentrations of acceptor-labeled peptide (ANT) (Fig. 2, A and B) were analyzed as indicated in Equation 1 (see “Experimental procedures) to determine the mole fraction of actin containing bound ANT (XDA) (Fig. 2A). Concentration–response of two known myosin-binding drugs, OM and Myk 461, did not affect actin–ANT FRET (Fig. 5C) This control experiment further indicates the structural specificity of the identified hit compounds for actin. Functional effects of the 10 hit compounds on actin-activated myosin ATPase (Fig. 6) were measured in a concentration-dependent manner. Compounds that affected the anisotropy decreased the erythrosine probe lifetime, suggesting that that changes in actin’s flexibility are associated with changes in the probe environment, increasing exposure to the quenching effect of solvent oxygen The changes in both anisotropy and phosphorescence lifetime were roughly proportional to changes in FRET efficiency (Fig. 8, C and D), suggesting that the compound-induced changes in FRET are associated with changes in actin structural dynamics. The compounds most effective in their effects on correlations between actin dynamics and the mechanism of activation of myosin ATPase [10, 22, 23]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
