UVM Theses and Dissertations
Format:
Print
Author:
Decarreau, Justin
Dept./Program:
Biochemistry
Year:
2011
Degree:
PhD
Abstract:
Directed force and motion are characteristics needed for all living organisms. Diffusion processes are· slow and non-specific and for organisms that are more complex, such as vertebrates, directed transport of specific cargoes are essential to life. Muscle cells are comprised mainly of actin tracks and myosin motors. The myosin molecule is an elongated structure that contains a long C-terminal tail domain used for filament fonnation, and an N-tenninal globular domain that contains binding sites for actin and ATP. By undergoing a cyclic interaction with actin and coupled with the hydrolysis of ATP and release of reaction products ADP and inorganic phosphate, myosin is able to produce force and motion within the cell.
Myosin molecules from many different sources are comprised of similar structur.e as well as similar enzymatic ATPase cycles. The basic enzymatic cycle for a myosin molecule comprises the following steps, a tightly bound actomyosin complex is disrupted by ATP binding, this causes a conformational change in the myosin molecule that leads to its dissociation from actin. Hydrolysis of ATP occurs when myosin is detached from actin, followed by rebinding of myosin to actin involving a slow isomerization of the myosin products complex that leads to strong actin binding. The slow isomerization is linked to phosphate release and force production, this step is followed by a faster step of ADP release returning the cycle to the beginning and a strongly bound actomyosin complex.
The enzymatic cycle is tightly linked to structural changes in the myosin molecule as nucleotide-sensing elements within the myosin nucleotide-binding pocket propagate small conformational changes into large confonnational changes in distal parts of the myosin motor. One region that undergoes a large scale structural change is the lever arm domain, a long single a-helical rod that acts in a ratchet-like to manner to cause a 100 Å displacement of the myosin molecule during a single round of ATP hydrolysis. Further confonnational changes are observed in the nucleotide-binding pocket and the actin-binding site, both of which show an open-to-closed motion depending on the nucleotide state of the motor.
One step in this cycle that is of particular importance is the ADP release step as this has been shown to regulate the sliding velocity of the actomyosin complex and therefore dictates the shortening velocity of a muscle. There are confonnational changes associated with ADP release observed in smooth muscle myosin such as an open-closed transition of the nucleotide-binding pocket as well as an ADP induced rotation of the lever arm that is unique to slower myosin motors. The conformational changes associated with ADP release in smooth muscle myosin are not well understood and are the focus of the studies presented in this dissertation.
Using tryptophan fluorescence as a signal the conformational changes of smooth muscle myosin during the ADP release step are monitored. We show for the first time an isofonn specific difference in the properties of a surface exposed loop that regulates nucleotide release from the motor. We also reveal properties of the open-to-closed transition of the active site of myosinin theADP bound state.
Myosin molecules from many different sources are comprised of similar structur.e as well as similar enzymatic ATPase cycles. The basic enzymatic cycle for a myosin molecule comprises the following steps, a tightly bound actomyosin complex is disrupted by ATP binding, this causes a conformational change in the myosin molecule that leads to its dissociation from actin. Hydrolysis of ATP occurs when myosin is detached from actin, followed by rebinding of myosin to actin involving a slow isomerization of the myosin products complex that leads to strong actin binding. The slow isomerization is linked to phosphate release and force production, this step is followed by a faster step of ADP release returning the cycle to the beginning and a strongly bound actomyosin complex.
The enzymatic cycle is tightly linked to structural changes in the myosin molecule as nucleotide-sensing elements within the myosin nucleotide-binding pocket propagate small conformational changes into large confonnational changes in distal parts of the myosin motor. One region that undergoes a large scale structural change is the lever arm domain, a long single a-helical rod that acts in a ratchet-like to manner to cause a 100 Å displacement of the myosin molecule during a single round of ATP hydrolysis. Further confonnational changes are observed in the nucleotide-binding pocket and the actin-binding site, both of which show an open-to-closed motion depending on the nucleotide state of the motor.
One step in this cycle that is of particular importance is the ADP release step as this has been shown to regulate the sliding velocity of the actomyosin complex and therefore dictates the shortening velocity of a muscle. There are confonnational changes associated with ADP release observed in smooth muscle myosin such as an open-closed transition of the nucleotide-binding pocket as well as an ADP induced rotation of the lever arm that is unique to slower myosin motors. The conformational changes associated with ADP release in smooth muscle myosin are not well understood and are the focus of the studies presented in this dissertation.
Using tryptophan fluorescence as a signal the conformational changes of smooth muscle myosin during the ADP release step are monitored. We show for the first time an isofonn specific difference in the properties of a surface exposed loop that regulates nucleotide release from the motor. We also reveal properties of the open-to-closed transition of the active site of myosinin theADP bound state.