Rubin, John E.2010-05-122010-05-122010-04-21https://hdl.handle.net/11299/62054Additional contributor: Joseph M. Autry; David D. Thomas (faculty mentor).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.en-USCollege of Biological SciencesDepartment of Biochemistry, Molecular Biology and BiophysicsPresentation