A fluorescence energy transfer method for analyzing protein oligomeric structure

Application to phospholamban

Ming Li, Laxma G. Reddy, Roberta Bennett, Norberto D. Silva, Larry Jones, David D. Thomas

Research output: Contribution to journalArticle

118 Citations (Scopus)

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 get 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°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 Å. 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.

Original languageEnglish
Pages (from-to)2587-2599
Number of pages13
JournalBiophysical Journal
Volume76
Issue number5
StatePublished - 1999

Fingerprint

Energy Transfer
Fluorescence
Proteins
Sarcoplasmic Reticulum
phospholamban
Coloring Agents
Membranes
Protein Subunits
Lipid Bilayers
Membrane Lipids
Point Mutation
Sodium Dodecyl Sulfate
Detergents
Amines
Adenosine Triphosphatases
Electrophoresis
Polyacrylamide Gel Electrophoresis
Phosphorylation

ASJC Scopus subject areas

  • Biophysics

Cite this

Li, M., Reddy, L. G., Bennett, R., Silva, N. D., Jones, L., & Thomas, D. D. (1999). A fluorescence energy transfer method for analyzing protein oligomeric structure: Application to phospholamban. Biophysical Journal, 76(5), 2587-2599.

A fluorescence energy transfer method for analyzing protein oligomeric structure : Application to phospholamban. / Li, Ming; Reddy, Laxma G.; Bennett, Roberta; Silva, Norberto D.; Jones, Larry; Thomas, David D.

In: Biophysical Journal, Vol. 76, No. 5, 1999, p. 2587-2599.

Research output: Contribution to journalArticle

Li, M, Reddy, LG, Bennett, R, Silva, ND, Jones, L & Thomas, DD 1999, 'A fluorescence energy transfer method for analyzing protein oligomeric structure: Application to phospholamban', Biophysical Journal, vol. 76, no. 5, pp. 2587-2599.
Li, Ming ; Reddy, Laxma G. ; Bennett, Roberta ; Silva, Norberto D. ; Jones, Larry ; Thomas, David D. / A fluorescence energy transfer method for analyzing protein oligomeric structure : Application to phospholamban. In: Biophysical Journal. 1999 ; Vol. 76, No. 5. pp. 2587-2599.
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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 get 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°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 {\AA}. 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.",
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