Abstract
Phospholamban (PLB) oligomerization, quaternary structure, and sarco(endo)plasmic reticulum calcium ATPase (SERCA) binding were quantified by fluorescence resonance energy transfer (FRET) in an intact cellular environment. FRET between cyan fluorescent protein-PLB and yellow fluorescent protein-PLB in AAV-293 cells showed hyperbolic dependence on protein concentration, with a maximum efficiency of 45.1 +/- 1.3%. The observed FRET corresponds to a probe separation distance of 58.7 +/- 0.5A(,) according to a computational model of intrapentameric FRET. This is consistent with models of the PLB pentamer in which cytoplasmic domains fan out from the central bundle of transmembrane helices. An I40A mutation of PLB did not alter pentamer conformation but increased the concentration of half-maximal FRET (K(D)) by >4-fold. This is consistent with the previous observation that this putatively monomeric mutant still oligomerizes in intact membranes but forms more dynamic pentamers than wild type PLB. PLB association with SERCA, measured by FRET between cyan fluorescent protein-SERCA and yellow fluorescent protein-PLB, was increased by the I40A mutation without any detectable change in probe separation distance. The data indicate that the regulatory complex conformation is not altered by the I40A mutation. A naturally occurring human mutation (L39Stop) greatly reduced PLB oligomerization and SERCA binding and caused mislocalization of PLB to the cytoplasm and nucleus. Overall, the data suggest that the PLB pentamer adopts a "pinwheel" shape in cell membranes, as opposed to a more compact "bellflower" conformation. I40A mutation decreases oligomerization and increases PLB binding to SERCA. Truncation of the transmembrane domain by L39Stop mutation prevents anchoring of the protein in the membrane, greatly reducing PLB binding to itself or its regulatory target, SERCA.
Highlights
Transfer efficiency measurements of FRET standard constructs showed that this photobleaching protocol obtained absolute FRET efficiency measurements in agreement with nondestructive techniques, such as 3-cube FRET and FLIM [23, 24, 31]
A bimolecular complex always shows a linear relationship between acceptor bleaching and donor dequenching (Fig. 4B), multiple acceptors per donor can result in sublinear dependence of donor fluorescence on acceptor fluorescence (Fig. 1C), because energy transfer within the complex is maintained even after some of the acceptors have been bleached
We examined PLB pentamer FRET over a range of protein concentrations and obtained several informative parameters from a hyperbolic fit of the data (Fig. 1D)
Summary
For CFP-YFP energy transfer, this distance is 49.2 Å [22], making these probes well suited to studying PLB pentamer and regulatory complex conformation. We examined PLB pentamer FRET over a range of protein concentrations and obtained several informative parameters from a hyperbolic fit of the data (Fig. 1D).
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