Interdomain fluorescence resonance energy transfer in SERCA probed by cyan-fluorescent protein fused to the actuator domain

Deborah L. Winters, Joseph M. Autry, Bengt Svensson, David D. Thomas

Research output: Contribution to journalArticle

31 Scopus citations

Abstract

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 [Ca2+] (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 Å between probes on the two domains. An increase of [Ca2+] 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 Å. 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.

Original languageEnglish (US)
Pages (from-to)4246-4256
Number of pages11
JournalBiochemistry
Volume47
Issue number14
DOIs
StatePublished - Apr 8 2008

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