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PDBsum entry 1xnu
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Signaling protein
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PDB id
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1xnu
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References listed in PDB file
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Key reference
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Title
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Phospholamban pentamer quaternary conformation determined by in-Gel fluorescence anisotropy.
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Authors
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S.L.Robia,
N.C.Flohr,
D.D.Thomas.
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Ref.
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Biochemistry, 2005,
44,
4302-4311.
[DOI no: ]
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PubMed id
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Abstract
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We measured in-gel fluorescence anisotropy of phospholamban (PLB) labeled with
the biarsenical fluorophore FlAsH at three different sites on the cytoplasmic
domain. The 6 kDa monomer bands of FlAsH-tetracysPLB showed high anisotropy (r =
0.29), reflecting null homotransfer and low mobility (S = 0.85) on the
nanosecond time scale of the FlAsH fluorescence lifetime. 30 kDa bands
(pentameric PLB) within the same lanes exhibited low anisotropy, suggesting
intrapentameric fluorescence energy homotransfer between PLB subunits. FlAsH
labels positioned at residue -6, 5, or 23 showed a graduated pattern of
fluorescence depolarization corresponding to resonance energy transfer radii of
46 +/-2, 38 +/- 4, and <25 A, respectively. Pentamer anisotropy increased
with heating or fluorescence photobleaching toward a maximum value similar to
that determined for monomeric PLB. Fluorescence resonance energy heterotransfer
was also observed in vitro and in vivo within PLB pentamers colabeled with FlAsH
and the biarsenical fluorophore ReAsH. In vitro heterotransfer efficiencies were
graduated by labeling position, in harmony with homotransfer results. The
calculated transfer radii compare favorably to distances predicted by a computer
molecular model of the phospholamban pentamer constructed from NMR solution
structures. The data support a helical pinwheel model for the PLB pentamer, in
which the cytoplasmic domains bend sharply outward from the central bundle of
helices.
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