Hypertrophic cardiomyopathy (HCM) is a symptomatic affliction due to mutations in cardiac contractile proteins. We compared a highly penetrant mutation in cardiac myosin, M493I, with wild type (WT) using an advanced optical trap assay capable of quantifying kinetic rates including actomyosin re-attachment. Kinetic changes in both the actomyosin attachment and detachment rates suggest that the equilibrium between a conformation termed the Interacting Head Motif (IHM) and freely available myosin heads is disturbed in M493I. This type of disruption has been hypothesized to lead to the toxic hypertrophy observed in patients.
Optical tweezers have greatly impacted the development of mechanobiology by enabling high precision sub-piconewton measurements of mechanical forces developed by force generating proteins, called molecular motors, at the single-molecule level. Molecular motors, such as kinesins hydrolyze ATP to generate force (10 pN) and transport in a directional manner intracellular cargoes along cytoskeletal filamentous tracks called microtubules. The force developed by kinesins have been mainly studied using the “single-bead” assay, where an optically trapped bead is pulled by a bead-attached kinesin molecule as it steps along a surface immobilized microtubule. This assay, besides forces parallel to the long axis of the filament on which the kinesin processes, forces perpendicular to the filament due to the bead interacting with the underlying microtubule. These perpendicular forces, which cannot be directly measured, can accelerate the detachment of the molecular motor from its filamentous track. An alternative approach is the “three-bead” assay, in which the vertical force component is minimized, and the total opposing force is mainly parallel to the microtubule. Experiments with kinesin sho
Imprecision in protein positioning and instrument dead time have hampered efforts to measure macromolecular association rates in optical trap assays. Here, we combine several technical improvements to the three-bead optical trap assay, including precise protein deposition, enhanced stage stability by feedback, and improved data filtering. These enhancements allow us to precisely and reliably detect interactions between cardiac heavy meromyosin (cHMM) and actin and quantify attachment and reattachment rates. These studies providing insights into strain-dependence of the power stroke and a proposed transition from super-relaxed (SRX) to disordered relaxed (DRX) states, which is thought to be disrupted in human hypertrophic cardiomyopathy.
We assembled an ultra-fast infrared optical trapping system to detect mechanical events that occur less than a millisecond
after a ligand binds to its filamentous substrate, such as myosin undergoing its 5 – 10 nm working stroke after actin binding.
The instrument is based on the concept of Capitanio et al.1, in which a polymer bead-actin-bead dumbbell is held in two
force-clamped optical traps. A force applied by the traps causes the filament to move at a constant velocity as
hydrodynamic drag balances the applied load. When the ligand binds, the filament motion stops within 100 μs as the total
force from the optical traps is transferred to the attachment. Subsequent translations signal active motions, such as the
magnitude and timing of the motor’s working stroke. In our instrument, the beads defining the dumbbell are held in
independent force clamps utilizing a field-programmable gate array (FPGA) to update the trap beam positions at 250 kHz.
We found that in our setup, acousto-optical deflectors (AODs) steering the beams were unsuitable for this purpose due to
a slightly non-linear response in the beam intensity and deflection angle vs. the AOD ultra-sound wavelength, likely caused
by low-amplitude standing acoustic waves in the deflectors. These aberrations caused instability in the force feedback
loops leading to artefactual ~20 nm jumps in position. This type of AOD non-linearity has been reported to be absent in
electro-optical deflectors (EODs)2. We demonstrate that replacement of the AODs with EODs improves the performance
of our instrument. Combining the superior beam-steering capability of the EODs, force acquisition via back-plane
interferometry, and the dual high-speed FPGA-based feedback loops, we smoothly and precisely apply constant loads to
study the dynamics of interactions between biological molecules such as actin and myosin.
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