Phospholamban pentamer quaternary conformation determined by in-gel fluorescence anisotropy.
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.
Robia
Seth L
SL
Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, Minnesota 55455, USA.
Flohr
Nicole C
NC
Thomas
David D
DD
D001152:Arsenicals / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003545:Cysteine / Q000235:genetics; D003593:Cytoplasm / Q000235:genetics / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D016650:Fluorescein-5-isothiocyanate / Q000031:analogs & derivatives* / Q000378:metabolism; D005452:Fluoresceins / Q000378:metabolism; D005454:Fluorescence Polarization / Q000379:methods; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009942:Organometallic Compounds / Q000378:metabolism; D010078:Oxazines / Q000378:metabolism; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary / Q000235:genetics; D012710:Serum Albumin, Bovine / Q000378:metabolism; D000099211:Phospholamban
D001152:Arsenicals; D002135:Calcium-Binding Proteins; D003545:Cysteine; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins; D005456:Fluorescent Dyes; D009942:Organometallic Compounds; D010078:Oxazines; D012710:Serum Albumin, Bovine; D000099211:Phospholamban; C113276:4',5'-bis(1,3,2-dithioarsolan-2-yl)fluorescein; C454115:ReAsH-EDT2 compound; C039217:fluorescein isothiocyanate bovine serum albumin