Phospholamban oligomerization, quaternary structure, and sarco(endo)plasmic reticulum calcium ATPase binding measured by fluorescence resonance energy transfer in living cells

Eileen M. Kelly, Zhanjia Hou, Julie B C Bossuyt, Donald M Bers, Seth L. Robia

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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 ± 0.5 Å, 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.

Original languageEnglish (US)
Pages (from-to)12202-12211
Number of pages10
JournalJournal of Biological Chemistry
Volume283
Issue number18
DOIs
StatePublished - May 2 2008
Externally publishedYes

Fingerprint

Reticulum
Oligomerization
Fluorescence Resonance Energy Transfer
Calcium-Transporting ATPases
Cells
Mutation
Conformations
phospholamban
Codonopsis
Proteins
Membranes
Cell membranes
Fans
Membrane Proteins
Cytoplasm

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Molecular Biology

Cite this

@article{ec012da1774949c8aba722cec83c6961,
title = "Phospholamban oligomerization, quaternary structure, and sarco(endo)plasmic reticulum calcium ATPase binding measured by fluorescence resonance energy transfer in living cells",
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 ± 0.5 {\AA}, 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.",
author = "Kelly, {Eileen M.} and Zhanjia Hou and Bossuyt, {Julie B C} and Bers, {Donald M} and Robia, {Seth L.}",
year = "2008",
month = "5",
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doi = "10.1074/jbc.M707590200",
language = "English (US)",
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pages = "12202--12211",
journal = "Journal of Biological Chemistry",
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TY - JOUR

T1 - Phospholamban oligomerization, quaternary structure, and sarco(endo)plasmic reticulum calcium ATPase binding measured by fluorescence resonance energy transfer in living cells

AU - Kelly, Eileen M.

AU - Hou, Zhanjia

AU - Bossuyt, Julie B C

AU - Bers, Donald M

AU - Robia, Seth L.

PY - 2008/5/2

Y1 - 2008/5/2

N2 - 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 ± 0.5 Å, 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.

AB - 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 ± 0.5 Å, 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.

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