Fluorescence Analysis of the Sarcolipin:SERCA Protein Complex

Rubin, John E.
2009-04-08
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Fluorescence Analysis of the Sarcolipin:SERCA Protein Complex

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2009-04-08

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We have used fluorescence resonance energy transfer (FRET) to identify physical interactions between the sarcoplasmic reticulum Ca-ATPase (SERCA) and one of its regulatory proteins, sarcolipin (SLN), in cardiac and skeletal muscle. The sarcoplasmic reticulum (SR) is an intracellular membrane network found in muscle cells whose function is to uptake, store, and release calcium. SERCA functions to transport calcium into the SR to induce muscle relaxation. Theoretical models predict that SLN monomers regulate SERCA by binding the SERCA transmembrane domain, but SLN monomers also self-associate to form oligomers. To test these models, we expressed fluorescent fusion proteins of SLN and SERCA in Sf21 insect cells using the baculovirus system. Quantitative binding stoichiometries were determined by FRET measurements using live cell microscopy on plates coated with mollusk “glue” protein. FRET results indicate that (1) SLN monomers self-associate to form dimers and (2) SLN monomers interact with SERCA to form a 1:1 heterocomplex. We propose that SLN monomers compete in equilibrium between SLN oligomerization and SERCA binding.

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Additional contributors: Joseph M. Autry; David D. Thomas (faculty mentor).

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This research was supported by the Undergraduate Research Opportunities Program (UROP). Additional support came from the National Institutes of Health grant no. GM27906.

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Rubin, John E.. (2009). Fluorescence Analysis of the Sarcolipin:SERCA Protein Complex. Retrieved from the University Digital Conservancy, https://hdl.handle.net/11299/50382.

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