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investigation. Single molecular TIRF microscopy was used to measure the translational diffusional coefficient of Alexa488 labeled monomeric PLB reconstituted into a supported lipid bilayer. The diffusional coefficient of monomeric PLB is 0.7 mm/s, which is consistent with its molecular weight. Timeresolved phosphorescence anisotropy of erythrosin iodoacetamide (ErIA) labeled SERCA in cardiac sarcoplasmic reticulum (SR) was measured with and without phosphorylation of PLB in presence of high and low Ca concentrations. Phosphorylation of PLB decreased the final anisotropy of ErIA labeled SERCA at low Ca, indicating decreased SERCA self-association. This supports the proposal that PLB inhibits SERCA by inducing SERCA-SERCA association, which is relieved by phosphorylation.