Date of Award

2019

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Graduate Group

Biochemistry & Molecular Biophysics

First Advisor

Yale E. Goldman

Second Advisor

E. M. Ostap

Abstract

Despite decades of research concerning the mechano-chemistry of the molecular motor myosin, significant questions remain regarding the mechano-chemical coupling of actin binding, lever arm rotation (the working stroke), and the release of inorganic phosphate. These processes are key to myosin’s ability to produce force, and all occur within just a few milliseconds, making it difficult to determine their properties. We have implemented and optimized an ultra-fast optical trapping technique which is able to precisely (<100 μs, <0.1 nm) observe the binding to and displacement of actin by myosin while myosin experiences a controllable load. With this technique, we find that human β-cardiac myosin initially interacts with actin in a short-lived state that has a lifetime of <1 ms but can support loads of > 4 pN. The working stroke of myosin occurs directly from this state at a rate of up to 5000 per second. This stroke can be rapidly reversed under resisting loads until phosphate is released from the myosin. After phosphate is released, high levels of free phosphate in solution (10 mM) can rebind to myosin, allowing the stroke to once again be reversed. We observe that the coupling between the working stroke and phosphate release can be disrupted by the addition of a small molecule drug, omecamtiv mecarbil (OM), which binds directly to cardiac myosin. This drug speeds up phosphate release, but inhibits the working stroke, diverting myosin off its canonical pathway and slowing detachment. We show that this slowed detachment is responsible for the beneficial effects of the drug in patients. This work represents one of the first direct, single molecule observations that the weak binding state of myosin is capable of sustaining pN-sized forces and is the first direct observation of the working stroke rate as a function of applied load. These findings provide strong evidence to place the working stroke before phosphate release in cardiac myosin’s biochemical cycle.

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