Event Title

Improved Methodology for Studying Actomyosin Structural Dynamics

Location

CSU Ballroom

Start Date

20-4-2015 10:00 AM

End Date

20-4-2015 11:30 AM

Student's Major

Chemistry and Geology

Student's College

Science, Engineering and Technology

Mentor's Name

Rebecca Moen

Mentor's Email Address

rebecca.moen@mnsu.edu

Mentor's Department

Chemistry and Geology

Mentor's College

Science, Engineering and Technology

Description

Myosin and its interaction with actin are responsible for force generation in muscle. The molecular level structural details of actomyosin dynamics coupled to contraction mechanism remain unclear. The catalytic domain of myosin contains its substrate-binding pocket, which binds ATP, and the actin-binding site termed the cleft. The actin-binding cleft is composed of the upper and lower 50 kDa domains. The cleft undergoes a structural change from open-to-closed upon force generation in muscle. This study aims to construct a myosin which will produce more accurate and discrete measurements of distance across the actin-binding cleft. The Dictyostelium (Dicty) myosin gene truncated at residue 761 and with no reactive cysteine residues was used as the base construct. Sitedirected mutagenesis was employed to introduce specific cysteine residues in the upper and lower 50 kDa domains of Dicty myosin II at positions [S387C.K391C] and [N541C.T545C]. Mutations were confirmed by DNA sequencing. The cysteine mutations produced will allow for future experiments in site-directed spin-labeling (SDSL) involving binding of a rigidly attached bifunctional spin label at these positions across the actin cleft. This new myosin construct will allow changes in myosin structural dynamics to be defined more precisely having large implications for future research on myosin and its actin-binding cleft.

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Apr 20th, 10:00 AM Apr 20th, 11:30 AM

Improved Methodology for Studying Actomyosin Structural Dynamics

CSU Ballroom

Myosin and its interaction with actin are responsible for force generation in muscle. The molecular level structural details of actomyosin dynamics coupled to contraction mechanism remain unclear. The catalytic domain of myosin contains its substrate-binding pocket, which binds ATP, and the actin-binding site termed the cleft. The actin-binding cleft is composed of the upper and lower 50 kDa domains. The cleft undergoes a structural change from open-to-closed upon force generation in muscle. This study aims to construct a myosin which will produce more accurate and discrete measurements of distance across the actin-binding cleft. The Dictyostelium (Dicty) myosin gene truncated at residue 761 and with no reactive cysteine residues was used as the base construct. Sitedirected mutagenesis was employed to introduce specific cysteine residues in the upper and lower 50 kDa domains of Dicty myosin II at positions [S387C.K391C] and [N541C.T545C]. Mutations were confirmed by DNA sequencing. The cysteine mutations produced will allow for future experiments in site-directed spin-labeling (SDSL) involving binding of a rigidly attached bifunctional spin label at these positions across the actin cleft. This new myosin construct will allow changes in myosin structural dynamics to be defined more precisely having large implications for future research on myosin and its actin-binding cleft.

Recommended Citation

Sinda, Daniel. "Improved Methodology for Studying Actomyosin Structural Dynamics." Undergraduate Research Symposium, Mankato, MN, April 20, 2015.
http://cornerstone.lib.mnsu.edu/urs/2015/poster_session_A/40