Molecular Motor-Based Assays for Altered Nanomechanical Function of Ca 2+ -Regulatory Proteins in Cardiomyopathies
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Molecular Motor-Based Assays for Altered Nanomechanical Function of Ca2+-Regulatory Proteins in Cardiomyopathies P. Bryant Chase1, Nicolas M. Brunet2, Goran Mihajlovic3, Peng Xiong3, and Stephan von Molnár3 1 Biological Science, Molecular Biophysics and Chemical & Biomedical Engineering, Florida State University, Tallahassee, FL, 32306-4370 2 Biological Science and Molecular Biophysics, Florida State University, Tallahassee, FL, 32306 3 Physics and MARTECH, Florida State University, Tallahassee, FL, 32306
ABSTRACT We propose that a thermo-electrical control system for rapid and reversible actuation of biomolecular motors and their partner filaments can also be used to study molecular mechanisms of cardiovascular diseases. We have previously used this device to evaluate the temperaturedependence of unregulated (absence of Ca2+-regulatory proteins tropomyosin, α-Tm, and troponin, Tn) actin filament sliding powered by ATP-hydrolyzing myosin motors. Assays using the thermo-electric controller can also be applied to regulated thin filaments (F-actin plus α-Tm and Tn) to obtain energetic parameters and functional correlates of structural stability at the level of single filaments. This allows us not only to examine Ca2+-dependent sliding of thin filaments, but also to test for altered function of clinically relevant mutations of cardiac myofilament proteins such as those identified in familial hypertrophic cardiomyopathy (FHC). INTRODUCTION Biomolecular motors hold tremendous potential for transport in nanoscale devices [1]. Myosin, which powers the transport of actin filaments by hydrolyzing ATP, is the biomolecular motor of muscle and can be adapted for use in a wide variety of artificial settings. Spatial control of actomyosin transport can be achieved through a variety of means, including guidance through chemically-defined patterning of functional motors [2-5] or actin [6], and through a combination of chemical and physical barriers [4,5]. Rapid and reversible, temporal control of motion can be achieved by varying temperature [7,8]. While actomyosin motility is remarkably stable over a wide range of temperatures when exposure to elevated temperatures is brief, a greater concern for functional stability is the issue of long-term storage [8]. Actomyosin transport can be accelerated not only by temperature, but also by substituting 2’-deoxy ATP for
ATP as the nucleotide substrate [9-11], or by adding Ca2+ and the Ca2+ regulatory proteins of striated muscle thin filaments, Tn and Tm [12,13]. Specific disease-related mutations of the Ca2+-regulatory proteins may be particularly interesting in this regard as some FHC mutations increase Ca2+-sensitivity of actomyosin function and may also further increase function at saturating [Ca2+] [14-16]. The mechanisms underlying these changes in function, and how these FHC mutations ultimately lead to hypertrophy of the heart and possibly sudden cardiac death, remain unknown [16-20]. METHODS Proteins were prepared for motility assays as described previously [7,13]. T
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