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.
Additional contributor: Joseph M. Autry; David D. Thomas (faculty mentor).
This project was funded by a grant to JER from the University of Minnesota Undergraduate Research Opportunity Program (UROP).
This project was funded by a grant to DDT from the National Institutes of Health (GM27906).
Rubin, John E..
Molecular Interactions of Sarcolipin and Phospholamban Using Fluorescence Resonance Energy Transfer (FRET) in Live Cells.
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