2-color calcium pump reveals closure of the cytoplasmic headpiece with calcium binding双色的钙泵显示关闭细胞质与钙结合盔.pdfVIP

2-Color Calcium Pump Reveals Closure of the Cytoplasmic Headpiece with Calcium Binding 1 1 1 2 2 1 Zhanjia Hou , Zhihong Hu , Daniel J. Blackwell , Tyler D. Miller , David D. Thomas , Seth L. Robia * 1 Department of Cell and Molecular Physiology, Loyola University Chicago, Maywood, Illinois, United States of America, 2 Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, Minnesota, United States of America Abstract The sarco(endo)plasmic reticulum calcium ATPase (SERCA) undergoes conformational changes while transporting calcium, but the details of the domain motions are still unclear. The objective of the present study was to measure distances between the cytoplasmic domains of SERCA2a in order to reveal the magnitude and direction of conformational changes. Using fluorescence microscopy of live cells, we measured intramolecular fluorescence resonance energy transfer (FRET) from a donor fluorescent protein fused to the SERCA N-terminus to an acceptor fluorescent protein fused to either the N-, P-, or transmembrane domain. The ‘‘2-color’’ SERCA constructs were catalytically active as indicated by ATPase activity in vitro and Ca uptake in live cells. All constructs exhibited dynamic FRET changes in response to the pump ligands calcium and thapsigargin (Tg). These FRET changes were quantified as an index of SERCA conformational changes. Intramolecular FRET decreased with Tg for the two N-domain fusion sites (at residue 509 or 576), while the P- (residue 661) and TM-domain (C- terminus) fusions showed increased FRET with Tg. The magnitude of the Tg-dependent conformational change was not decreased by coexpression of phospholamban (PLB), nor did PLB