Interdomain fluorescence resonance energy transfer in SERCA probed by cyan-fluorescent protein fused to the actuator domain.
We have used a biosynthetically incorporated fluorescent probe to monitor domain movements involved in ion transport by the sarcoendoplasmic reticulum Ca-ATPase (SERCA) from rabbit fast-twitch skeletal muscle. X-ray crystal structures suggest that the nucleotide-binding (N) and actuator (A) domains of SERCA move apart by several nanometers upon Ca binding. To test this hypothesis, cDNA constructs were created to fuse cyan-fluorescent protein (CFP) to the N terminus of SERCA (A domain). This CFP-SERCA fluorescent fusion protein retained activity when expressed in Sf21 insect cells using the baculovirus system. Fluorescence resonance energy transfer (FRET) was used to monitor the A-N interdomain distance for CFP-SERCA selectively labeled with fluorescein isothiocyanate (FITC) at Lys 515 in the N domain. At low [Ca (2+)] (E2 biochemical state), the measured FRET efficiency between CFP (donor in A domain) and FITC (acceptor in N domain) was 0.34 +/- 0.03, indicating a mean distance of 61.6 +/- 2.0 A between probes on the two domains. An increase of [Ca (2+)] to 0.1 mM (E1-Ca biochemical state) decreased the FRET efficiency by 0.06 +/- 0.03, indicating an increase in the mean distance by 3.0 +/- 1.2 A. Quantitative molecular modeling of dual-labeled SERCA, including an accurate calculation of the orientation factor, shows that the FRET data observed in the absence of Ca is consistent with the E2 crystal structure, but the increase in distance (decrease in FRET) induced by Ca is much less than predicted by the E1 crystal structure. We conclude that the E1 crystal structure does not reflect the predominant structure of SERCA under physiological conditions in a functional membrane bilayer.
Winters
Deborah L
DL
Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, Minnesota 55455, USA.
Autry
Joseph M
JM
Svensson
Bengt
B
Thomas
David D
DD
D000818:Animals; D002460:Cell Line; D003198:Computer Simulation; D018360:Crystallography, X-Ray; D019286:Endopeptidase K / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D015870:Gene Expression; D049452:Green Fluorescent Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D020836:Protein Structure, Quaternary; D011817:Rabbits; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D018411:Spodoptera
C485182:Cyan Fluorescent Protein; D049452:Green Fluorescent Proteins; D019286:Endopeptidase K; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases