GST Pulldown Analysis: Identification of Interactions Between CPβ1/CPβ2 and Novel Proteins
Location
CSU 253/254/255
Start Date
13-4-2004 12:45 PM
End Date
13-4-2004 2:45 PM
Student's Major
Biological Sciences
Student's College
Science, Engineering and Technology
Mentor's Name
Marilyn Hart
Mentor's Department
Biological Sciences
Mentor's College
Science, Engineering and Technology
Description
Actin capping protein (CP), a heterodimer composed of a and p subunits, binds the barbed ends of actin filaments and regulate actin's specific binding affinities. Similar forms of the p subunit (βl, β2, and β3), encoded by one gene, have been identified. Alternative splicing confers variation in the C terminus region of the β1 and β2 isoforms. Although the N terminus of CP β proteins are necessary for actin binding, the role of the variable C terminus regions remains undefined. Prior research, preformed by the primary investigator of this study, suggests that the β1 isoform is essential for attaching actin filaments to the Z-line of heart tissue, and the β2 isoform organizes actin at the intercalated discs of heart tissue. Thus, CP contributes to cardiac function and accordingly, mutations in CP become suspects in the causation of heart disease. We hypothesize that CP β1 and β2 interact with novel proteins at their C terminal ends and these interactions define their different roles in organizing actin filaments in the myocardium. The purpose of this study is to identify novel proteins that interact with the β1and β2 subunits using Glutathione-S-Transferase (GST) pulldown analysis. The gene encoding the β1and β2 proteins has been inserted into the expression vector pGEX-3X, allowing the generation of GST-β1 and GST- β2 fusion proteins. The fusion protein is currently being used to bind proteins interacting with the β subunits using the GST as an anchor. If successful, the GST- β complexes will identify proteins interacting with the CP β subunits.
GST Pulldown Analysis: Identification of Interactions Between CPβ1/CPβ2 and Novel Proteins
CSU 253/254/255
Actin capping protein (CP), a heterodimer composed of a and p subunits, binds the barbed ends of actin filaments and regulate actin's specific binding affinities. Similar forms of the p subunit (βl, β2, and β3), encoded by one gene, have been identified. Alternative splicing confers variation in the C terminus region of the β1 and β2 isoforms. Although the N terminus of CP β proteins are necessary for actin binding, the role of the variable C terminus regions remains undefined. Prior research, preformed by the primary investigator of this study, suggests that the β1 isoform is essential for attaching actin filaments to the Z-line of heart tissue, and the β2 isoform organizes actin at the intercalated discs of heart tissue. Thus, CP contributes to cardiac function and accordingly, mutations in CP become suspects in the causation of heart disease. We hypothesize that CP β1 and β2 interact with novel proteins at their C terminal ends and these interactions define their different roles in organizing actin filaments in the myocardium. The purpose of this study is to identify novel proteins that interact with the β1and β2 subunits using Glutathione-S-Transferase (GST) pulldown analysis. The gene encoding the β1and β2 proteins has been inserted into the expression vector pGEX-3X, allowing the generation of GST-β1 and GST- β2 fusion proteins. The fusion protein is currently being used to bind proteins interacting with the β subunits using the GST as an anchor. If successful, the GST- β complexes will identify proteins interacting with the CP β subunits.
