Event Title

Oxidation Effects the Myosin Motor Domain Function and Structure

Location

CSU Ballroom

Start Date

11-4-2017 10:00 AM

End Date

11-4-2017 11:30 AM

Student's Major

Chemistry and Geology

Student's College

Science, Engineering and Technology

Mentor's Name

Rebecca Moen

Mentor's Department

Chemistry and Geology

Mentor's College

Science, Engineering and Technology

Description

This research examines the functional and structural effects of oxidative modification in Dicty myosin II in the motor domain. Myosin has been shown to be a target of oxidative stress. Reactive oxygen species target specific amino acids within contractile proteins leading to site directed oxidative modifications. Oxidative stress of muscle proteins is associated with aging and chronic diseases that induce muscle wasting. The goal was to obtain molecular level information on the myosin-actin relationship to understand the effects of myosin oxidation and muscle dysfunction using site-directed mutagenesis (SDM), molecular modeling, enzyme kinetics, site-direct spin- labeling and electron paramagnetic resonance (EPR). The structural focus was on the force- generating domain of myosin, specifically the relay helix, which undergoes a large structural change with contraction. SDM was used to introduce new cysteine sites to monitor structural changes in the relay helix. Labels bind by disulfide linkage to amino acid residues at i and i+4 on an alpha helix at sites 492 and 496, and 639 and 643 in the myosin primary sequence. The function of this mutated myosin was tested using an actin-activated ATPase assay to confirm that neither mutagenesis nor spin-labeling affected myosin's function. The oxidized labeled-myosin actin- activated ATPase activity showed a 3-fold decrease in activity compared to the unoxidized labeled myosin. Results for changes in myosin structural dynamics based on EPR data is yet to be determined although simulations of the data suggest a change will be detectable. These results provide knowledge on protein oxidation effects with potential therapeutic significance.

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Apr 11th, 10:00 AM Apr 11th, 11:30 AM

Oxidation Effects the Myosin Motor Domain Function and Structure

CSU Ballroom

This research examines the functional and structural effects of oxidative modification in Dicty myosin II in the motor domain. Myosin has been shown to be a target of oxidative stress. Reactive oxygen species target specific amino acids within contractile proteins leading to site directed oxidative modifications. Oxidative stress of muscle proteins is associated with aging and chronic diseases that induce muscle wasting. The goal was to obtain molecular level information on the myosin-actin relationship to understand the effects of myosin oxidation and muscle dysfunction using site-directed mutagenesis (SDM), molecular modeling, enzyme kinetics, site-direct spin- labeling and electron paramagnetic resonance (EPR). The structural focus was on the force- generating domain of myosin, specifically the relay helix, which undergoes a large structural change with contraction. SDM was used to introduce new cysteine sites to monitor structural changes in the relay helix. Labels bind by disulfide linkage to amino acid residues at i and i+4 on an alpha helix at sites 492 and 496, and 639 and 643 in the myosin primary sequence. The function of this mutated myosin was tested using an actin-activated ATPase assay to confirm that neither mutagenesis nor spin-labeling affected myosin's function. The oxidized labeled-myosin actin- activated ATPase activity showed a 3-fold decrease in activity compared to the unoxidized labeled myosin. Results for changes in myosin structural dynamics based on EPR data is yet to be determined although simulations of the data suggest a change will be detectable. These results provide knowledge on protein oxidation effects with potential therapeutic significance.

Recommended Citation

Ott, Rachel. "Oxidation Effects the Myosin Motor Domain Function and Structure." Undergraduate Research Symposium, Mankato, MN, April 11, 2017.
http://cornerstone.lib.mnsu.edu/urs/2017/poster-session-A/32