Modelling the actin-myopalladin complex using crosslinking mass spectrometry

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Meuli, Rose Marie
Ajiboye, Oluwatosin
Beck, Moriah R.
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Meuli, Rose Marie, Ajiboye, Oluwatosin and Beck, Moriah R. Modelling the actin-myopalladin complex using crosslinking mass spectrometry. -- Fyre in STEM Showcase, 2023.

Myopalladin is an actin-binding protein that has recently been discovered in striated muscle tissue. Like its family members myotilin and palladin, myopalladin contains immunoglobin (Ig) domains that participate in actin binding. Myopalladin acts to stabilize actin and limit actin polymerization, but mutations in myopalladin have been linked to cardiomyopathy. A model of myopalladin's structure would provide a better understanding of this protein's role in cardiomyopathy. Therefore, the Beck lab has conducted an experiment to determine the structure of the myopalladin-actin complex and compare that structure with the better-known familial proteins palladin and myotilin. Like palladin, myopalladin's Ig3 domain has been identified by the Beck Lab as the minimum site required for binding to actin. The Ig3 domain of myopalladin was first crosslinked with filamentous (F-) actin using various chemical crosslinkers to create covalent bonds between the two proteins, thus trapping them in the complex. The crosslinked proteins were separated and visualized using an SDS-PAGE gel and DMTMM was determined to result in the most crosslinks between Ig3 and F-actin. The SDS-PAGE gel bands containing the Ig3-actin complex were then sent for crosslinking mass spectrometry (XL-MS) analysis. The results from the XL-MS will provide us with precise fragment masses that can be used to determine which amino acids from each protein are in close proximity. This data will then be used to determine a structural model of myopalladin via the use of programs such as P2-Link and HADDOCK. Once the myopalladin-actin complex has been modelled, the actin-binding sites can also be located. This information will provide insight into the actin-myopalladin interaction, which can then be used in comparison to palladin and myotilin to increase overall knowledge of these Ig-domain-containing proteins. Overall, the data collected in this experiment has the potential to be crucial for the better understanding of cardiomyopathy.

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Poster and abstract presented at the FYRE in STEM Showcase, 2023.
Research project completed at the Department of Chemistry and Biochemistry.
Wichita State University
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FYRE in STEM 2023
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