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 pharmacologi-cally 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.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 12288-12298 |
| Number of pages | 11 |
| Journal | Journal of Biological Chemistry |
| Volume | 293 |
| Issue number | 31 |
| DOIs | |
| State | Published - Aug 3 2018 |
Bibliographical note
Funding Information:This work was supported by National Institutes of Health Grants R01 HL129814, R01 AR32961, and R37 AG26160 (to D. D. T.) and R42 DA037622 (to Fluorescence Innovations, Inc.; subcontract to the University of Minne-sota). D. D. T. holds equity in, and serves as President of, Photonic Pharma LLC. This relationship has been reviewed and managed by the University of Minnesota. Photonic Pharma had no role in this study. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Funding Information:
This work was supported by National Institutes of Health Grants R01 HL129814, R01 AR32961, and R37 AG26160 (to D. D. T.) and R42 DA037622 (to Fluorescence Innovations, Inc.; subcontract to the University of Minnesota). D. D. T. holds equity in, and serves as President of, Photonic Pharma LLC. This relationship has been reviewed and managed by the University of Minnesota. Photonic Pharma had no role in this study. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Fluorescence experiments were performed at the Biophysical Technology Center, University of Minnesota. We thank Samantha Yuen for technical assistance, Dr. Ben Binder for critical review, and Octavian Cornea for preparation of the manuscript.
Publisher Copyright:
© 2018 Guhathakurta et al.