期刊
BIOCHEMISTRY
卷 47, 期 47, 页码 12448-12456出版社
AMER CHEMICAL SOC
DOI: 10.1021/bi8014289
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资金
- National Institutes of Health [HL64031]
- Genomics:GTL program [45701]
- Office of Biological and Environmental Research
- U.S. Department of Energy
- Pacific Northwest National Laboratory
- Department of Energy by Battelle Memorial Institute [DE-AC05-76RLO 1830]
Calcium-dependent domain movements of the actuator (A) and nucleotide (N) domains of the SERCA2a isoform. of the Ca-ATPase were assessed using constructs containing engineered tetracysteine binding motifs, which were expressed in insect High-Five cells and subsequently labeled with the biarsenical fluorophore 4',5'-bis(1,3,2-dithioarsolan-2-yl)fluorescein (FlAsH-EDT2). Maximum catalytic function is retained in microsomes isolated from High-Five cells and labeled with FlAsH-EDT2. Distance measurements using the nucleotide analog 2',3'-O-(2,4,6-trinitrophenyl) adenosine 5'-triphosphate (TNP-ATP), which acts as a fluorescence resonance energy transfer (FRET) acceptor from FlAsH, identify a 2.4 angstrom increase in the spatial separation between the N- and A-domains induced by high-affinity calcium binding; this structural change is comparable to that observed in crystal structures. No significant distance changes occur across the N-domain between FlAsH and TNP-ATP, indicating that calcium activation induces rigid body domain movements rather than intradomain conformational changes. Calcium-dependent decreases in the fluorescence of FlAsH bound, respectively, to either the N- or A-domains indicate coordinated and noncooperative domain movements, where both A- and N-domains display virtually identical calcium dependencies (i.e., K-d = 4.8 +/- 0.4 mu M). We suggest that occupancy of a single high-affinity calcium binding site induces the rearrangement of the A- and N-domains of the Ca-ATPase to form an intermediate state, which facilitates phosphoenzyme formation from ATP upon occupancy of the second high-affinity calcium site.
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