Browsing by Author "Rubin, John E."

Now showing 1 - 2 of 2
  • Thumbnail Image
    Item
    Fluorescence Analysis of the Sarcolipin:SERCA Protein Complex
    (2009-04-08) Rubin, John E.
    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.
  • Thumbnail Image
    Item
    Molecular Interactions of Sarcolipin and Phospholamban Using Fluorescence Resonance Energy Transfer (FRET) in Live Cells
    (2010-04-21) Rubin, John E.
    We have used fluorescence resonance energy transfer (FRET) microscopy to measure the binding affinities of four protein interactions in muscle to elucidate the binding events that occur during the formation of the SERCA super-inhibitory complex. Calcium is transported into the sarcoplasmic reticulum (SR) during muscle relaxation by the sarcoplasmic reticulum Ca- ATPase (SERCA), which is separately regulated by two transmembrane proteins, sarcolipin (SLN) and phospholamban (PLB). It has been proposed that when SERCA, SLN, and PLB are all expressed in the same muscle cell, the three proteins bind together in a super-inhibitory ternary complex, which decreases SERCA calcium transport by 50%. A key intermediate to this proposed ternary complex is the SLN:PLB heterocomplex. In my project, FRET microscopy was used to confirm the presence of the SLN:PLB heterocomplex and to directly quantitate the degree physical interaction between the two proteins in live cells. For comparison, FRET microscopy was also used to quantify SLN:SLN, SLN:SERCA, and PLB:SERCA interactions. Average FRET was directly calculated for each protein:protein interaction on a cell-to-cell basis. In addition, a Michaelis-Menten binding model and non-linear Hill fitting were used to calculate the dissociation constant for each protein interaction and the intrinsic distance between fluorescent probes. FRET results indicated that SLN and PLB form a low affinity heterodimer in cells with a distance of 4.6 nm between subunits. FRET results also show that SLN:SLN has the highest binding affinity of the four interactions while SERCA:SLN and SERCA:PLB have medium binding affinities relative to SLN:PLB and SLN:SLN. We propose that SLN and PLB first bind independently to SERCA and then bind to each other to induce the super-inhibitory SERCA ternary complex.