Expression Studies of Other Unconventional Myosins

National Heart, Lung, And Blood Institute

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Abstract

In collaboration with Philipp Kukura of Oxford University, we have used a light microscopy based interferometric scattering technique to examine the processive movement of myosin 5 HMM on actin. Using this technique we were able to image single, unlabeled molecules of myosin 5 HMM move along actin with a precision of a few nanometers. The molecule took 36 nm steps and moved at the same speed as previously reported for fluorescently-labeled myosin 5. By attaching a 20 nm gold particle to the amino-terminus we are able to measure the movement at sampling rates up to 1000 Hz and follow the movement of the unattached labeled myosin head. Interesting, even with a 20 nm gold particle attached the myosin moves at the same velocity as the unlabeled molecule. The unlabeled head does not freely diffuse as previously proposed, but rather spends most of its time in a fixed, off actin, position from which it occasionaly explores the forward actin binding sites. Simultaneous tracking of both heads revealed that consecutive steps follow identical paths to the same side of actin in a compass-like spinning motion demonstrating a symmetrical walking pattern. In collaboration with the lab of Tom Friedman we have examined the molecular properties of a myosin-15 bearing a deafness associated mutation. We have expressed and purified myosin-5B and have measured the ATPase and in vitro motility of this protein at NIH and have collaborated with Marco Capitanio's lab in Italy measuring the mechanical properties of this myosin in an optical trap.