A Fluorescence Energy Transfer Method for Analyzing Protein Oligomeric Structure: Application to Phospholamban

Abstract

We have developed a method using fluorescence energy transfer (FET) to analyze protein oligomeric structure. Two populations of a protein are labeled with fluorescent donor and acceptor, respectively, then mixed at a defined donor/acceptor ratio. A theoretical simulation, assuming random mixing and association among protein subunits in a ring-shaped homo-oligomer, was used to determine the dependence of FET on the number of subunits, the distance between labeled sites on different subunits, and the fraction of subunits remaining monomeric. By measuring FET as a function of the donor/acceptor ratio, the above parameters of the oligomeric structure can be resolved over a substantial range of their values. We used this approach to investigate the oligomeric structure of phospholamban (PLB), a 52-amino acid protein in cardiac sarcoplasmic reticulum (SR). Phosphorylation of PLB regulates the SR Ca-ATPase. Because PLB exists primarily as a homopentamer on sodium dodecyl sulfate polyacrylamide gel electrophoresis, it has been proposed that the pentameric structure of PLB is important for its regulatory function. However, this hypothesis must be tested by determining directly the oligomeric structure of PLB in the lipid membrane. To accomplish this goal, PLB was labeled at Lys-3 in the cytoplasmic domain, with two different amine-reactive donor/acceptor pairs, which gave very similar FET results. In detergent solutions, FET was not observed unless the sample was first boiled to facilitate subunit mixing. In lipid bilayers, FET was observed at 25 degrees C without boiling, indicating a dynamic equilibrium among PLB subunits in the membrane. Analysis of the FET data indicated that the dye-labeled PLB is predominantly in oligomers having at least 8 subunits, that 7-23% of the PLB subunits are monomeric, and that the distance between dyes on adjacent PLB subunits is about 10 A. A point mutation of PLB (L37A) that runs as monomer on SDS-PAGE showed no energy transfer, confirming its monomeric state in the membrane. We conclude that FET is a powerful approach for analyzing the oligomeric structure of PLB, and this method is applicable to other oligomeric proteins.

p62/Sequestosome-1 Associates with and Sustains the Expression of Retroviral Restriction Factor TRIM5α

“…Such a deviation from linearity is observed for multimeric complexes containing multiple FRET acceptors (5,22). The degree of curvature is related to the number of probes in the complex and the probe separation distance (25). When the average ratios of the measured fluorescence relative to the original fluorescence (F/F 0 ) for CFP-rhTRIM5␣ and YFP-p62 (Fig.…”

Modulation of physiological and pathological activities of lysozyme by biological membranes

“…(S27)-(S31)). This model allows one to determine the parameters of protein self-association, such as degree of oligomerization (n), the distance between monomers in protein assembly (R a ) and fraction of donors present in oligomers (X), from the results of time-resolved FRET studies [59]. However, when trying to approximate the experimental data by the above model we faced the problem of strong correlation between the calculated parameters.…”

Acute Inotropic and Lusitropic Effects of Cardiomyopathic R9C Mutation of Phospholamban

“…Probe separation distance (R) for the SERCA-PLB regulatory complex was calculated using the Förster equation (40), r ϭ (R O ) ((1/IE) Ϫ 1) 1/6 ), where R O is the Förster radius, and E is the measured FRET max . Intrapentameric probe separation distance was calculated from FRET max using a MatLab application (34), assuming a ring-shaped oligomer (41)(42)(43), with a subunit number of 5 (pentamer). The acceptor molar fraction was 0.89 for the WT-PLB pentamer and 0.92 for the R9C-PLB pentamer.…”