- Duchenne muscular dystrophy is a lethal muscle disease, caused by mutations in the gene encoding dystrophin, an actin-binding cytoskeletal protein. Absence of functional dystrophin results in muscle weakness and degeneration, eventually leading to cardiac and respiratory failure. Strategies to replace the missing dystrophin via gene therapy have been intensively pursued. However, the dystrophin gene is too large for current gene therapy approaches. Currently available micro-dystrophin constructs lack the actin-binding domain 2 and show decreased actin-binding affinity in vitro compared to full-length dystrophin.
- Cardiac myosin-binding protein C (cMyBP-C) interacts with actin and myosin to modulate cardiac muscle contractility. These interactions are disfavored by cMyBP-C phosphorylation. Heart failure patients often display decreased cMyBP-C phosphorylation, and phosphorylation in model systems has been shown to be cardioprotective against heart failure. Therefore, cMyBP-C is a potential target for heart failure drugs that mimic phosphorylation or perturb its interactions with actin/myosin. Here we have used a novel fluorescence lifetime-based assay to identify small-molecule inhibitors of actin-cMyBP-C binding.
- Myosins generate force and motion by precisely coordinating their mechanical and chemical cycles, but the nature and timing of this coordination remains controversial. We utilized a FRET approach to examine the kinetics of structural changes in the force-generating lever arm in myosin V. We directly compared the FRET results with single-molecule mechanical events examined by optical trapping. We introduced a mutation (S217A) in the conserved switch I region of the active site to examine how myosin couples structural changes in the actin- and nucleotide-binding regions with force generation.
- Actin's interactions with myosin and other actin-binding proteins are essential for cellular viability in numerous cell types, including muscle. In a previous high-throughput time-resolved FRET (TR-FRET) screen, we identified a class of compounds that bind to actin and affect actomyosin structure and function. For clinical utility, it is highly desirable to identify compounds that affect skeletal and cardiac muscle differently. Because actin is more highly conserved than myosin and most other muscle proteins, most such efforts have not targeted actin.
- Myosins are molecular motors that use a conserved ATPase cycle to generate force. We investigated two mutations in the converter domain of myosin V (R712G and F750L) to examine how altering specific structural transitions in the motor ATPase cycle can impair myosin mechanochemistry. The corresponding mutations in the human β-cardiac myosin gene are associated with hypertrophic and dilated cardiomyopathy, respectively. Despite similar steady-state actin-activated ATPase and unloaded in vitro motility–sliding velocities, both R712G and F750L were less able to overcome frictional loads measured in the loaded motility assay.
- 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.
- Many biological processes, including cell division, growth, and motility, rely on rapid remodeling of the actin cytoskeleton and on actin filament severing by the regulatory protein cofilin. Phosphorylation of vertebrate cofilin at Ser-3 regulates both actin binding and severing. Substitution of serine with aspartate at position 3 (S3D) is widely used to mimic cofilin phosphorylation in cells and in vitro. The S3D substitution weakens cofilin binding to filaments, and it is presumed that subsequent reduction in cofilin occupancy inhibits filament severing, but this hypothesis has remained untested.