Data on whole length myosin binding protein C stabilizes myosin S2 as measured by gravitational force spectroscopy.

Data presented in this article relates to the research article entitled "Whole length myosin binding protein C stabilizes myosin subfragment-2 (S2) flexibility as measured by gravitational force spectroscopy." (Singh et al., 2018) [1]. The data exhibits the purified skeletal myosin binding protein C (MyBPC) from rabbit back muscle was of slow skeletal type confirmed by chromatography and in unphosphorylated state based on its isoelectric point (pI) by chromatofocussing. The competitive enzyme linked immunosorbent assay (cELISA) data displayed the site specificity of polyclonal anti-S2 antibody to myosin S2. This polyclonal antibody binding site corresponds to a familial hypertrophic cardiomyopathy (FHC) point mutation hotspot on myosin S2 illustrated in a figure of compiled data.

Whole length myosin binding protein C stabilizes myosin S2 as measured by gravitational force spectroscopy.

The mechanical stability of the myosin subfragment-2 (S2) was tested with simulated force spectroscopy (SFS) and gravitational force spectroscopy (GFS). Experiments examined unzipping S2, since it required less force than stretching parallel to the coiled coil. Both GFS and SFS demonstrated that the force required to destabilize the light meromyosin (LMM) was greater than the force required to destabilize the coiled coil at each of three different locations along S2. GFS data also conveyed that the mechanical stability of the S2 region is independent from its association with the myosin thick filament using cofilaments of myosin tail and a single intact myosin. The C-terminal end of myosin binding protein C (MyBPC) binds to LMM and the N-terminal end can bind either S2 or actin. The force required to destabilize the myosin coiled coil molecule was 3 times greater in the presence of MyBPC than in its absence. Furthermore, the in vitro motility assay with full length slow skeletal MyBPC slowed down the actin filament sliding over myosin thick filaments. This study demonstrates that skeletal MyBPC both enhanced the mechanical stability of the S2 coiled coil and reduced the sliding velocity of actin filaments over polymerized myosin filaments.

Demonstrating the uses of the novel gravitational force spectrometer to stretch and measure fibrous proteins.

The study of macromolecular structure has become critical to the elucidation of molecular mechanisms and function. There are several limited, but important bioinstruments capable of testing the force dependence of structural features in proteins. Scale has been a limiting parameter on how accurately researchers can peer into the nanomechanical world of molecules, such as nucleic acids, enzymes, and motor proteins that perform life-sustaining work. Atomic force microscopy (AFM) is well tuned to determine native structures of fibrous proteins with a distance resolution on par with electron microscopy. However, in AFM force studies, the forces are typically much higher than a single molecule might experience. Optical traps (OT) are very good at determining the relative distance between the trapped beads and they can impart very small forces. However, they do not yield accurate absolute lengths of the molecules under study. Molecular simulations provide supportive information to such experiments, but are limited in the ability to handle the same large molecular sizes, long time frames, and convince some researchers in the absence of other supporting evidence. The gravitational force spectrometer (GFS) fills a critical niche in the arsenal of an investigator by providing a unique combination of abilities. This instrument is capable of generating forces typically with 98% or better accuracy from the femtonewton range to the nanonewton range. The distance measurements currently are capable of resolving the absolute molecular length down to five nanometers, and relative bead pair separation distances with a precision similar to an optical trap. Also, the GFS can determine stretching or uncoiling where the force is near equilibrium, or provide a graded force to juxtapose against any measured structural changes. It is even possible to determine how many amino acid residues are involved in uncoiling events under physiological force loads. Unlike in other methods where there is extensive force calibration that must precede any assay, the GFS requires no such force calibration. By complementing the strengths of other methods, the GFS will bridge gaps in understanding the nanomechanics of vital proteins and other macromolecules.