GROVE, T.J.*; MCFADDEN, L.A.; CHASE, P.B.; MOERLAND, T.S.; Florida State University: Characterization of Myosin from Fundulus heteroclitus by In Vitro Motility Assay

The killifish, Fundulus heteroclitus, has adapted to a widely fluctuating intertidal environment where these fish experience dramatic changes in temperature and salinity over the course of a tidal cycle. F. heteroclitus exploits this variable habitat and is widely distributed along the Atlantic coast of the United States from Maine to Georgia. We asked how physiological changes in temperature, ionic strength, and pH may affect interactions between myosin and F-actin, which power muscle contraction via ATP hydrolysis. We measured from in vitro motility assays (flow cells) the velocity of unregulated fluorescent actin filaments as they interacted with purified myosin from anaerobic skeletal muscle of F. heteroclitus from Woods Hole, MA. Conditions of a standard flow cell were 25�C, pH 7.4, and 32mM KCl; a single parameter was varied for each set of experiments. As temperature increased, actin motility significantly increased (p=0.007) and ranged from 0.13 + 0.02 &microm/sec at 11�C to 3.02 + 0.23 &microm/sec at 36�C. A Q_{10(11-36�C)} value of 3.55 was calculated from mean F-actin velocities. Actin motility was also measured over a range of ionic strengths from 16mM to 100mM KCl. Maximum velocity, 1.83 + 0.08 &microm/sec, was measured at 32mM KCl. Finally, motility significantly increased (p=0.043) within the range of pH 6.7 to 7.4, and the maximum velocity was measured at pH 7.4. Molecular studies to elucidate amino acid residues within the myosin heavy chain that may enable myosin to function over the potentially extreme environmental variability facing F. heteroclitus are underway. The molecular motor myosin and other proteins from organisms that are adapted to extreme environments may be useful in bio-nanotechnology devices. Supported by DARPA contract No. N66001-02-C-8030.